Trifluoro(pentafluoroethoxy)ethylene

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Four groups of male rats and female rats were exposed to concentrations of the test substance via whole-body inhalation for six hours per day for a total of 20 exposures over a four-week period. One-half of the control and high dose group animals were allowed to recover for a 14-day period.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Trifluoro(pentafluoroethoxy)ethylene

EC Number:

234-018-7

EC Name:

Trifluoro(pentafluoroethoxy)ethylene

Cas Number:

10493-43-3

Molecular formula:

C4F8O

IUPAC Name:

1,1,2-trifluoro-2-(1,1,2,2,2-pentafluoroethoxy)ethene

Test material form:

gas

Details on test material:

- Purity: 99%

Test animals

Species:

rat

Strain:

other: Crl:CD® (SD)BR

Details on species / strain selection:

Rats have historically been used in safety evaluation studies for inhalation toxicity testing. The Crl:CD®(SD)BR rat was selected based on consistently acceptable health status and on extensive experience with the strain at the testing laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks old
- Weight at study initiation: Males: 235-268 g Females: 174-207 g
- Housing: During quarantine and the test period, rats were housed singly in suspended, stainless steel, wire-mesh cages. Male and female rats were located on separate cage racks.
- Diet: ad libitum except during exposure and fasting prior to necropsy
- Water: ad libitum except during exposure
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22-23°C
- Humidity: 41-65%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: gas

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: All exposure chambers were constructed of stainless steel and glass (NYU style) with a nominal internal volume of approximately 150 liters. Directly inside the chamber inlet, a baffle was positioned to provide uniform distribution of the gas within the chamber.
- Method of holding animals in test chamber: Rats were placed in stainless steel wire mesh cages (sexes separate) and placed inside the exposure chamber.
- Source and rate of air: Gas atmospheres of the test substance were generated by metering the test substance gas from a cylinder, located in a heated water bath, or from a heated liquid trap. Both the cylinder and liquid trap were located in a remote safety enclosure for flammable gases. The test substance gas flowed through 1/4 inch (O.D.) stainless steel or Teflon® tubing and a rotometer prior to entry into the mixing flask.
- Method of conditioning air: Houseline air added to the mixing flask carried the test substance/air mixture into the exposure chamber. The chamber concentration of the test substance was controlled by regulating the flow of test substance gas through the rotometer.
- Temperature, humidity, pressure in air chamber: Chamber temperature was targeted at 22±2°C. Chamber relative humidity was targeted at 50±10%. Chamber oxygen concentration was targeted to at least 19%. Airflow, temperature, and relative humidity were monitored continually and recorded at 15-minute intervals during each exposure. Percent oxygen was measured with a Biosystems Oxygen Monitor and recorded two times during each exposure.
- Air change rate: 12 air changes/hour
- Treatment of exhaust air: Nitrogen was added to the high chamber exhaust and chamber atmospheres were exhausted directly into the fume hoods.

TEST ATMOSPHERE
- Brief description of analytical method used: The atmospheric concentration of the test substance was determined by gas chromatography at approximately 20-minute intervals during each six-hour exposure. Gas samples were drawn by vacuum pump from representative areas of the chamber where rats were exposed. Chamber atmosphere samples were directly injected into a gas chromatograph equipped with a flame ionization detector for analysis of the test substance concentration. All samples were chromatographed isothermally at 85°C on a 3% OV-17 Chromosorb column. The atmospheric concentration of the test substance was determined from a standard curve derived from gas standards. The gas standards were prepared prior to each exposure by injecting known volumes of test substance gas into Tedlar® bags that contained known volumes of air.

VEHICLE
- Composition of vehicle: Houseline air

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytically determined mean concentrations ± SEM of the test substance in the exposure chambers targeted to 500, 3000, or 18000 ppm were 500 ± 2.1, 3000 ± 10, and 18000 ± 57 for the 20 exposures. Daily mean concentrations were consistent from day- to-day throughout the study. The mean daily concentrations were within ±7% of the targeted exposure concentrations. During the 10th exposure, brief excursions in atmospheric concentration occurred in all test chambers during the first hour of exposure. The chamber concentrations measured during this period ranged from 31 to 920 ppm in the 500 ppm chamber, 260 to 2800 ppm in the 3000 ppm chamber, and 5500 to 17000 ppm in the 18000 ppm chamber. The excursions were due to the test substance condensing in the stainless steel lines. The problem with the generation system was corrected and the exposures continued without additional excursions. The brief excursions in chamber concentration were considered not to have adversely affected the results of the study. The analytically determined concentrations were considered acceptable for evaluating the toxicity of the test substance at the selected targeted concentrations.

Duration of treatment / exposure:

Six hours per day

Frequency of treatment:

Five days per week for a total of 20 exposures over a four-week period (weekends excluded)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 ppm

Remarks:

±2.1 ppm

Doses / concentrations 2

Dose / conc.:

3 000 ppm

Remarks:

± 10 ppm

Doses / concentrations 3

Dose / conc.:

18 000 ppm

Remarks:

±57 ppm

No. of animals per sex per dose:

10 rats each for the control and high-concentration groups, and five rats each for the low- and intermediate-concentration

Control animals:

yes

Details on study design:

- Dose selection rationale: A range finding experiment was conducted in male and female rats exposed whole-body for six hours/day for four consecutive days to 18000 ppm of test substance. During these exposures, rats exhibited a diminished response to an alerting stimulus and a diminished activity level when compared to baseline evaluations prior to each exposure. Clinical signs of toxicity were limited to irregular respiration in one male and one female rat immediately after the second and third exposures, respectively. Rats had slight body-weight losses during the exposure period. For the four-week study, the highest concentration level of the test substance was targeted at 18000 ppm. This concentration was selected after considering the findings of the aforementioned information and the safety issues associated with generating the test substance gas in the 150 L exposure chambers. The high level exposure concentration at 18000 ppm is 60% of the lower explosive limit (30000 ppm) of the test substance.

Examinations

Observations and examinations performed and frequency:

BODY WEIGHT & CLINICAL OBSERVATIONS
- During the exposure phase of the study, all rats were weighed and individually observed for clinical signs twice per week before exposure and were individually observed for clinical signs after each six-hour exposure. Group clinical observations of rats visible from the front of the chamber were made during exposures. In addition, rats visible from the front of the chamber were checked for an alerting response to an auditory stimulus two times during each exposure and all rats were observed immediately after each exposure. During the 14-day recovery period, all remaining rats in the control and high-concentration groups were weighed and individually observed for clinical signs twice per week.

FOOD CONSUMPTION AND FOOD EFFICIENCY
- The amount of food consumed by each rat was determined weekly during the study. From these determinations and body weight data, mean individual daily food consumption and food efficiency were calculated.

HAEMATOLOGY
- Anaesthetic used for blood collection: Yes (Light carbon dioxide)
- Animals fasted: Yes
- Parameters checked in table No.1 were examined.

CLINICAL CHEMISTRY
- Animals fasted: Yes
- Parameters checked in table No.2 were examined.

URINALYSIS
- Parameters checked in table No.3 were examined.

Sacrifice and pathology:

GROSS PATHOLOGY & HISTOPATHOLOGY
Five animals/group were examined at day 26 on test. Following 14 days of recovery, the remaining rats in the control and high concentration groups were necropsied. The liver, kidneys, lungs, brain, adrenals, testes, and ovaries were weighed at necropsy. Each rat was given a complete gross examination and representative samples of the following tissues were saved: liver, kidneys, lungs, heart, skeletal muscle, spleen, aorta, brain, spinal cord, stomach, duodenum, jejunum, ileum, pancreas, cecum, colon, rectum, mesenteric lymph node, mandibular lymph node, exorbital lacrimal glands, salivary gland, thymus, adrenal glands, sciatic nerve, pituitary gland, thyroid gland, parathyroid glands, trachea, oesophagus, pharynx/larynx, Zymbal's glands, eyes, skin, mammary gland (female), ovaries, uterus, vagina, prostate, seminal vesicles, urinary bladder, testes, epididymides, femur (including knee joint), sternum, bone marrow, nose, and gross lesions.
All tissues were fixed in 10% neutral buffered formalin except testes, epididymides, and eyes which were fixed in Bouin's solution. The lungs were weighed then inflated with 10% neutral buffered formalin at necropsy. Tissues from rats in the 18000 ppm and control groups that were sacrificed at the end of the exposure period were processed, embedded in paraffin, cut at a nominal thickness of five micrometers, stained with haematoxylin and eosin (H&E), and examined with a microscope. The nose, pharynx/larynx, liver, kidneys, and lungs from rats in the 500 and 3000 ppm groups were also processed to slides and examined microscopically. For the recovery animals, the kidneys were processed to slides and examined microscopically.

Statistics:

Descriptive statistics including mean, standard deviation, and standard error of the mean were used to summarize experimental data. Incidences of clinical observations were evaluated by the Cochran-Armitage test for trend. Mean body weights, body weight gains, food consumption, mean final body weights, and mean absolute and relative (to body weight and to brain weight) organ weights were statistically analyzed with a one-way analysis of variance (ANOVA). Pairwise comparisons between test and control groups (sexes separate) were made with the Dunnett's test. The Bartlett's test for homogeneity of variances and significance was performed on organ weight data. Except for the Bartlett's test (alpha = 0.005), all significance was judged at alpha = 0.05.
For clinical pathologic evaluations, ANOVA and Bartlett's tests were calculated for each sampling time. Dunnett's test was used to compare means from the control groups and each of the groups (sexes separate) exposed to the test substance. When the results of the Bartlett's test were significant (alpha = 0.005), the Kruskal-WaIIis test was employed and the Mann-Whitney U test was used to compare means from the control groups and each of the groups exposed to the test substance. Significance was judged at alpha = 0.05.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During exposures, rats visible from the front of the exposure chamber were observed for clinical signs of toxicity and for their reaction to an alerting stimulus. Rats were inadvertently not checked for clinical signs of toxicity during the first exposure; however, their reaction to an alerting stimulus was evaluated. This oversight did not affect the results of the study. No clinical signs of toxicity were evident in male or female rats in the 500 or 3000 ppm groups during exposures, and these rats, when compared to controls, exhibited a normal response to an alerting stimulus. In general, rats in the 18000 ppm group displayed a normal response to an alerting stimulus. These rats did, however, display diminished responses during the 8th, 13th, 14th, and 16th exposures. No other clinical signs of toxicity were evident in these rats during exposures.

Immediately after exposures, no clinical signs of toxicity attributable to the test substance were evident. Moreover, rats visible from the front of the exposure chamber displayed a normal response to an auditory stimulus. Rats from the 18000 ppm group did show statistically significant increased incidences of stained and wet fur, nasal discharge (females), and alopecia (males). These findings, however, were considered not to be adverse. No clinical signs of toxicity were evident in rats from the 18000 ppm group during the recovery period.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no compound-related mortalities on the study. One female rat from the 18000 ppm group was sacrificed in extremis on day three of the recovery period due to eye trauma sustained during orbital blood collection. All remaining rats survived to their scheduled termination.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male rats in the 18000 ppm exposure group had significantly lower mean body weights throughout both the exposure and recovery phases of the study. No additional statistically significant differences in mean body weights were observed in male rats from the other groups. Compared to controls, no statistically significant differences in mean body weights were found in any test group of female rats during the exposure period. However, mean body weights of female rats in the 18000 ppm group were consistently lower compared to controls during the exposure period. Although not statistically significant, the lower body weights in this group were considered compound-related. Female rats in the 18000 ppm group had significantly lower mean body weights compared to controls during the first week of recovery. Although not statistically significant, the mean body weights of female rats from the 18000 ppm group were lower than controls at the end of the recovery period.

Male and female rats exposed to 18000 ppm test substance had significantly lower mean body weight gains compared to controls at various intervals during the exposure period. In addition, mean body weight gains in male rats in the 18000 ppm group were significantly lower than controls at the conclusion of the exposure period. No additional toxicologically important differences in mean body weight gains were observed in male or female rats during the exposure period. Male and female rats from the 18000 ppm groups had higher mean body weight gains compared to controls during the recovery phase.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption for male rats in the 18000 ppm group were significantly lower compared to controls during the exposure period (days 1-22). No additional statistically or toxicologically important findings on food indices were seen in male or female rats during the study.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Food efficiency for male rats in the 18000 ppm group were significantly lower compared to controls during the exposure period (days 1-22). Female rats in the 18000 ppm group exhibited significantly lower mean food efficiency compared to controls during the first week of exposure and during the overall exposure period (days 1- 22). During the recovery period, the females had significantly higher mean food efficiency compared to controls. No additional statistically or toxicologically important findings on food indices were seen in male or female rats during the study.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Male and female rats in the 18000 ppm groups had decreased mean total leukocyte counts (WBC, statistically significant) at the end of the exposure period. Leukocyte counts were decreased primarily because of decreases in lymphocytes (statistically significant) and, less importantly, because of decreases in neutrophils (statistically significant in 18000 ppm males) or monocytes (statistically significant in 18000 ppm females). The causal relationship between test substance-exposure and the leukocytic alterations is equivocal. Decreased circulating lymphocyte counts could be a primary, test substance­ related effect resulting in decreased production of lymphocytes. However, absence of morphological alterations in spleen, lymph nodes, thymus and bone marrow does not indicate that such a primary, treatment-related effect had occurred. Furthermore, in rodents, the most common cause of decreased lymphocyte counts is increased release of endogenous glucocorticoids. This latter mechanism represents a secondary effect associated with "stress" in which endogenous glucocorticoids cause redistribution of lymphocytes from the circulation into lymphoid tissue. Repeated inhalation exposure to high concentrations of an offensive chemical could cause this type of stress response. For these reasons, the leukocytic changes in 18000 ppm males and females were not considered to be biologically adverse. Likewise, mildly decreased mean total leukocyte and lymphocyte counts in 3000 ppm females (not statistically significant) were not considered to be important for the same reasons.

Other statistically significant haematology findings were observed in male and female rats, but they were not considered to be toxicologically important for the following reasons:
•Significant changes is some parameters were not considered to be test substance­ related because the mean values did not exhibit a dose-response relationship. These changes included decreased mean Hb in 3000 ppm males and decreased mean monocyte count in 500 ppm females.
•Significantly increased mean RBC in 18000 ppm females was not considered to be biologically adverse because mild increases in RBC are biologically inconsequential. Rather, it is decreases in RBC which may indicate biological adversity.
•Significantly decreased mean MCH in 3000 and 18000 ppm females at the end of the exposure period and decreased mean MCV in 18000 ppm females at the 2-week recovery sampling time were not considered to be biologically adverse because there were no relevant changes in the indicators of circulating erythrocyte mass (RBC, Hb, Ht) in these groups.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Test substance-related or biologically adverse clinical chemical changes did not occur during this study. Some statistically significant clinical chemical findings were observed in male and female rats, but they were not considered to be important for the following reasons:

•Significant decreases in mean ALT ( 18000 ppm males and females at the end of the exposure period), SDH (18000 ppm males at the end of the recovery period), or bilirubin concentration ( 18000 ppm females at the end of the exposure period) were not considered to be adverse because decreases in serum enzyme activity or bilirubin concentration are not relevant to organ injury or dysfunction. Rather, it is increases in serum enzyme activity or bilirubin concentration which may indicate liver toxicity.
•Significantly decreased mean triglyceride concentration in 18000 ppm males at the end of the exposure period was not considered to be adverse. Mild decreases in triglyceride concentration are biologically inconsequential. Likewise, significantly decreased mean triglyceride concentration in 18000 ppm males at the end of the recovery period was not considered to be adverse, because adverse changes did not occur at the end of the exposure period. Also, the control group mean value at the end of the recovery period was unusually high.
•Significantly decreased mean total protein concentrations in 18000 ppm males and females at the end of the recovery period were not considered to be important because similar changes did not occur at the end of the exposure period. Also, there were no changes in serum albumin or globulin concentration.
•Significantly decreased mean BUN (18000 ppm males and females at the end of the exposure period), creatinine ( 18000 ppm females at the end of the exposure period, and 18000 males and females at the end of the recovery period) were not considered to be adverse because decreases in BUN or creatinine concentration are not relevant to organ injury or dysfunction. Conversely, it is increases in serum BUN or creatinine concentration which may indicate kidney dysfunction.
•In males, mild, statistically significant decreases in phosphate, calcium, and potassium concentration were not considered to be adverse because the magnitude of the changes was biologically inconsequential.
•Significantly increased chloride concentration in 18000 ppm males at the end of the recovery period was not considered to be important because similar changes did not occur at the end of the exposure period.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the exposure period, male and female rats in the 18000 ppm groups had unequivocal urine analytical changes which indicated diuresis. In these groups, urine volume was increased and osmolality was decreased (statistically significant). These changes indicate impaired ability to concentrate urine and they are likely associated with the renal tubular lesions which were observed during histopathological evaluation. Therefore, the urine analytical changes in the 18000 ppm groups were considered to be biologically adverse. Interestingly, these changes were not accompanied by increased BUN or creatinine, which would have indicated a decreased glomerular filtration rate. At the end of the recovery period, urine volume was slightly increased and osmolality was slightly decreased in 18000 ppm male and female rats (the osmolality change was statistically significant in males). Therefore, kidney dysfunction appeared to persist through the 2-week recovery period.

In the male and female 3000 ppm groups, urine osmolality was decreased (statistically significant in males) and volume was mildly increased (not statistically significant). These changes suggested that mild, compound related diuresis may have occurred at 3000 ppm. However, since there was no histopathological evidence for renal tubular injury, the changes at 3000 ppm were not considered to be biologically adverse.

Significantly increased urine pH in 18000 ppm males at the end of the exposure period may have been test substance-related, however, the magnitude of the change was biologically inconsequential.

Male and female rats in the 3000 and 18000 ppm groups had increased mean urine fluoride excretion (statistically significant). In the 18000 ppm groups the changes were unequivocal at the end of the exposure period and at the end of the recovery period. In the 3000 ppm male group at end of the exposure period, the mean urine fluoride excretion value exhibited a dose-response relationship and the change was likely test substance­related. In the 3000 ppm female group at the end of the exposure period, the mean urine fluoride excretion value was significantly higher than the control value but it was slightly less than the mean value for the 500 ppm group. However, one rat in the 500 ppm group had a urine fluoride excretion value which was much higher than other animals in the group and this caused the mean value to be unusually high. Elimination of this rat's urine fluoride excretion value causes the mean in the 500 ppm group to be less than the mean of the 3000 ppm group. Therefore, in spite of the apparent lack of a dose-response relationship for the female 3000 ppm mean urine fluoride excretion value, the change was likely test substance-related. The changes in urine fluoride excretion rate in 3000 and 18000 ppm males and females indicate that metabolism of the test substance causes release of fluoride ion. This was expected and was not considered to be biologically adverse.

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 end of the exposure period, significantly increased mean absolute and relative (to body and brain) kidney weights in the 18000 ppm females were test substance related. Significantly increased absolute kidney weight in the 3000 ppm female group was spurious since there was no significant increase in kidney weight relative to brain weight or microscopic change. Absolute lung weights and lung weights relative to body and brain weights were significantly increased in males and females at 18000 ppm. However, these lung weight changes were not associated with correlative microscopic changes and thus their biological significance is uncertain. Adrenal gland weights in the 18000 ppm females were also spurious and not biologically important, since there were no microscopic changes observed.

At the end of recovery period, significantly increased mean relative (to body) kidney weight in the 18000 ppm females was partly due to relatively lighter body weight and was considered not to be biologically important. Similarly, significant increases in mean relative (to body) kidney, lung, and brain weights in the 18000 ppm males were the results of body weight effects in that group.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related microscopic changes were observed in the kidneys in the 18000 ppm males and females at the end of the exposure period, and in the 18000 ppm females at the end of the recovery period. At the end of the exposure period, the renal changes were tubular degeneration/necrosis/regeneration which were observed in all five females and in five males. The lesions occurred in the outer stripe of the outer medulla, and were characterized by tubular epithelial cell degeneration, necrosis, sloughing, and basophilia and megalocytosis of regenerative cells. Mitotic figures were few. Female rats had more accentuated lesions than male rats. The renal changes appeared to be reversible. One 18000 ppm recovery female rat had very slight tubular cell degeneration and a few megalocytes in a few tubules. The other three only had a few megalocytes, residual from the exposure period, while the 18000 ppm males appeared fully recovered. In these three recovery females, renal tubular megalocytosis was listed as a separate diagnosis to illustrate the ongoing reparative response and near recovery of the lesions.

One 18000 ppm female, which was sacrificed in extremis on day three of the recovery period, had ocular lesions and optic nerve necrosis resulting from orbital blood collection, in addition to kidney changes. All other changes noted in microscopic incidence tables are known to occur spontaneously in rats and were not present in a dose response fashion in either incidence or severity. Thus, these observations are considered spontaneous and not associated with exposure to the test substance.

Effect levels

Key result

Dose descriptor:

NOEC

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

18 000 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Relevant for humans:

yes

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the no-observed-effect level for repeated exposure to the test substance in male and female rats was 3000 ppm.

Executive summary:

Four groups of male rats and four groups of female rats (ten rats each for the control and high-concentration groups, and five rats each for the low- and intermediate-concentration groups) were exposed to concentrations of the test substance targeted to 0, 500, 3000, or 18000 ppm. Animal exposures were whole-body for six hours per day, five days per week for a total of 20 exposures over a four-week period (weekends excluded). At the end of the exposure period, blood and urine samples were collected for clinical analyses, and five rats per group were sacrificed for pathologic examination. All remaining rats in the control and high-concentration groups were allowed to recover for a 14-day period. At the end of the recovery period, blood and urine samples were collected for clinical evaluation, and all surviving rats were sacrificed for pathologic examination.

The analytically determined mean concentrations ± SEM of the test substance in the exposure chambers targeted to 500, 3000, or 18000 ppm were 500 ± 2.1, 3000 ± 10, and 18000 ± 57 ppm for the 20 exposures. The daily mean exposure chamber temperatures ranged from 23-27°C. The daily mean relative humidity in the exposure chambers was between 34-57%, and the oxygen concentration was 20-21%. The daily mean chamber airflow ranged from 33 to 35 L/min.

Rats exposed to 18000 ppm had significantly lower mean body weights, body weight gains, food consumption, and food efficiency during the exposure period when compared to controls.

There were no compound-related mortalities in the study. During exposure, rats exposed to 18000 ppm had diminished response to an alerting stimulus. This effect was not observed in the other test groups. No other clinical signs of toxicity attributed to the test substance were observed during the study.

Clinical pathology evaluations revealed male and female rats in the 3000 and 18000 ppm groups had urine analytical changes which indicated diuresis, and the changes were evident at the end of the exposure period and at the end of the 2-week recovery period. In these groups, the changes which indicated a diuretic effect were increased urine volume and decreased urine osmolality. The changes in the 18000 ppm groups were considered to be compound related and biologically adverse because they were accompanied by histopathological lesions in the renal tubules. In the 3000 ppm groups, the changes may have been compound related but they were not considered to be biologically adverse because they were not accompanied by renal tubular lesions. Urine fluoride excretion was increased in 3000 and 18000 ppm males and females, but the changes were attributed to metabolism of the test substance and considered not to be biologically adverse. Male and female rats in the 18000 ppm groups had decreased mean total leukocyte, lymphocyte, neutrophil, and monocyte counts at the end of the exposure period. Female rats in the 3000 ppm group had decreased mean total leukocyte and lymphocyte counts. These changes were not accompanied by histopathological changes in lymphopoietic or hematopoietic organs. Most likely, the changes were due to redistribution of leukocytes due to stress and they were considered not to be biologically adverse.

Test substance-related statistically significant increases in organ weights occurred in mean absolute and relative kidney weights in female rats in the 18000 ppm group at the end of the exposure period. Microscopic effects in the kidneys of 18000 ppm males and females were zonal changes characterized by tubular epithelial degeneration/necrosis/regeneration in the outer stripe of the outer medulla of the kidneys. Slight residual kidney changes were noted in 18000 ppm females, but not males, after 14 days of recovery.

The no-observed-effect level (NOEL) for this study is defined as that dose which demonstrates the absence of adverse effects observed or measured at higher doses. The NOEL is equivalent to the no-observed-adverse-effect level (NOAEL) defined by the European Union. In this study, the test substance produced effects on body weight, organ weight, and food consumption, altered response to an alerting stimulus, clinical pathologic effects, and histologic effects in rats exposed at 18000 ppm. Under the conditions of this study, the no-observed-effect level for repeated exposure to the test substance in male and female rats was 3000 ppm.