2-Butene, 1,1,1,4,4,4-hexafluoro-, (2Z)-

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment. This was the most recent 90-day study and the dose selection was specifically designed to evaluate concentrations between the NOAEL of 1500 ppm (10064 mg/m3) and the LOAEL of 10000 ppm (67100 mg/m3) in a previous 90-day inhalation study. The dose selection in this study clearly identified the appropriate NOAEL/LOAEL for use in risk assessments. Although data provided have a report year after 2009, the study was performed to fulfill needs required by other governmental registrations and/or product stewardship purposes. This study was not performed to specifically fulfill an information requirement under REACH, but since the test data were already available they were provided as part of the REACH submission.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

Remarks:

The study was conducted according to the guideline in effect at the time of study conduct.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Deviations:

no

Remarks:

The study was conducted according to the guideline in effect at the time of study conduct.

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)

Deviations:

no

Remarks:

The study was conducted according to the guideline in effect at the time of study conduct.

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.5395 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Remarks:

The study was conducted according to the guideline in effect at the time of study conduct.

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Remarks:

The study was conducted according to the guideline in effect at the time of study conduct.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(2Z)-1,1,1,4,4,4-hexafluorobut-2-ene

EC Number:

700-651-7

Cas Number:

692-49-9

Molecular formula:

C4H2F6

IUPAC Name:

(2Z)-1,1,1,4,4,4-hexafluorobut-2-ene

Details on test material:

- Purity: >99.9%

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6 weeks
- Weight at study initiation: Mean group body weight range: 149.1 - 152.1 g (males); 143.2 - 144.9 g (females)
- Housing: group housed in solid bottom caging (2-3 rats per cage) with bedding and enrichment. Each cage rack contained only animals of one sex.
- Diet (e.g. ad libitum): ad libitum, except during exposures
- Water (e.g. ad libitum): ad libitum, except during exposures
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26ºC
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

clean 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 350 L. A tangential feed inside the chambers promoted uniform chamber distribution of the test atmosphere. The chamber volume was chosen so that the total body volume of the test animals did not exceed 5% of the chamber volume.
- Method of holding animals in test chamber: animals were placed in a wire-mesh module and exposed, whole-body
- Source and rate of air: Houseline high-pressure air, metered to the flask by a mass flow controller, carried the resulting atmosphere into a filtered dilutional air stream leading to the exposure chamber. The dilution air supply was metered by a mass flow controller.
- System of generating particulates/aerosols: Chamber atmospheres were generated by flash evaporation of the test substance in air with a heated, round-bottom, flash evaporation flask. The test substance was metered into the flask with a pump equipped with pistons. The flask was heated to approximately 175°C to vaporize the test substance. Houseline high-pressure air, metered to the flask by a mass flow controller, carried the resulting atmosphere into a filtered dilutional air stream leading to the exposure chamber. The dilution air supply was metered by a mass flow controller. General and dilutional air flows and the heating mantles were all monitored by the Camile Inhalation Toxicology Automated Data System (CITADS). Chamber concentrations of test substance were controlled by varying either the test substance feed rate to the flask or the amount of generational or dilutional air supply.
- Temperature, humidity, flow in air chamber: 20 – 24°C, 22 – 50% humidity, 60 L/min which provided 10 air changes per hour
- Treatment of exhaust air: exhausted into a fume hood

TEST ATMOSPHERE
- Brief description of analytical method used: During each exposure the atmospheric vapour concentration of the test substance was determined by gas chromatography at approximately 30 minute intervals. The air-control chamber was sampled once a week. Samples of the chamber atmosphere were continually drawn from the exposure chamber and directly injected into a gas chromatograph (GC) equipped with a gas sample valve and a flame-ionization detector. Samples were chromatographed isothermally at 80°C on a fused silica glass column.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The mean, analytically determined vapour concentrations for the 66 rat exposures were 0 ± 0, 3000 ± 8.4, 4000 ± 12, 5000 ± 10 and 7500 ± 8.3 ppm

Duration of treatment / exposure:

90 days

Frequency of treatment:

6 hours/day; 5 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 3000, 4000, 5000 and 7500 ppm
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 3000 ± 8.4, 4000 ± 12 , 5000 ± 10 and 7500 ± 8.3 ppm
Basis:
analytical conc.

No. of animals per sex per dose:

10/sex/concentration, for which 5 animals/sex/concentration were designated for micronucleus testing.

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: A previous subchronic inhalation toxicity study with the test substance reported a NOAEL of 1500 ppm where the next highest exposure concentration was 10000 ppm. Since there was a large gap between the NOAEL and the next highest concentration in the previous study, the objective of this study was to assess the potential subchronic toxicity of the test substance in rats to provide a more precise no-observed-adverse-effect-level (NOAEL) as well as evaluate the ability of the test substance to cause DNA damage as assessed with the micronucleus assay. The micronucleus assay evaluated the ability of the test substance to induce an increase in the frequency of micronucleated polychromatic erythrocytes (MNPCEs) in rat peripheral blood cells following inhalation exposure. An increase in this frequency relative to negative controls indicates that a test substance induced chromosome and/or spindle damage in erythroblasts.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Day 0 and weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0 and weekly thereafter

FOOD CONSUMPTION and FOOD EFFICIENCY: Yes
- Food consumption: The amount of food consumed by each animal over each weighing interval was determined by weighing each feeder at the beginning and end of the interval and subtracting the final weight and the amount of spillage from the feeder during the interval from the initial weight. From these measurements, mean daily food consumption over the interval was determined. From the food consumption and body weight data, the mean daily food efficiency was calculated.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretest and Week 12
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 14
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table No.1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 14
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table No.2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Week 14
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table No.2 were examined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table No. 3)

HISTOPATHOLOGY: Yes (see Table No. 3)

Microscopic examination was conducted on all tissues collected from the control and high concentration (7500 ppm) rats sacrificed by design following the 90-day exposure (10 rats/sex/exposure concentration). Potential target organs (nose, teeth and femur/knee joint), identified following the examination of control and high-exposure tissues, were examined from all rats. Sections from nose levels I, II, III and IV were examined from all the rats (sections from nose levels I and II contain the anterior nose and the incisors; sections from nose levels III and IV contain the posterior nose and the molars).

Other examinations:

MICRONUCLEUS EVALUATION:
- Time schedule for examinations: Test Day 3, Week 4, and Week 14
- See study DI.K1.90Day.InhVap.MicrNuc.R.D-17453-785-1.KD in Section 7.6.2 for details on Micronucleus test

Statistics:

See Materials and Methods below

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: The only clinical observations made during this study were sporadic incidences of hair loss or superficial wounds. There were no test substance-related causes of death in this study. All rats survived until their scheduled sacrifice.

BODY WEIGHT AND WEIGHT GAIN: Male rats exposed to 7500 ppm began demonstrating statistically significant reductions in body weight when compared to the air-exposed control group on test day 14 which continued until the end of the exposure period. At the end of the exposure period, the mean body weights of male rats exposed to 7500 ppm was 16% lower than the mean body weight of the control group. After the first week of exposure, male rats exposed to 7500 ppm began demonstrating statistically significant reductions in body weight gains that occurred until test day 42. The overall (test day 0 – 91) mean body weight gain of male rats exposed to 7500 ppm was also statistically reduced when compared to the control group by 22%. The reduction in mean body weight and body weight gain in males exposed to 7500 ppm was considered test substance-related and adverse. Male rats exposed to 5000 ppm demonstrated a statistically significant reduction in body weight on test day 91; however, this difference was not considered adverse since it was less than 15% different than the air control value and there were only sporadic reductions in body weight gain over the 3 month exposure period. There were no statistically significant changes in the mean body weights in rats expose to either 4000 or 3000 ppm, when compared to the air control group. Only a transient reduction in body weight gain was observed in male rats exposed to 5000 ppm on test days 35-42, 49-56 and the overall period of 0-91 and in males exposed to 4000 ppm from test day 7-14. The NOAEL for body weight reduction in male rats was 5000 ppm. Female rats exposed to the test substance resulted in no statistically significant effects on mean body weight at any exposure level. Exposure of female rats to 7500 ppm resulted in a statistically reduced body weight gain on test day 0–7 and for the overall period of test day 0–91; however, these results are considered non-adverse since there were no effects on the mean body weight parameters. The NOAEL for body weight reduction in female rats was 7500 ppm.

FOOD CONSUMPTION and FOOD EFFICIENCY: Male rats exposed to 7500 ppm began demonstrating statistically significant reductions in mean daily food consumption on test day 7 which lasted over the entire exposure period, while only sporadic reductions in food efficiency were observed on test day 0-7 and 7-14 in this group. Since this reduction in food consumption correlated with the adverse reduction in mean body weights, this finding was considered test substance-related and adverse. Male rats exposed to 5000 ppm began demonstrating statistically significant reductions in food consumption starting on test day 28 that continued to the end of the exposure period; however, there were no changes in food efficiency. Therefore the observations in the 5000 ppm exposure group were considered non-adverse. Transient, statistically significant reductions in food consumption were observed in male rats exposed to 3000 ppm on test days 21–28, 70-77 and 77-84 with no observed changes in food efficiency. These changes observed in the 3000 ppm male group were also not concentration-dependent and were considered spurious and non-adverse. Female rats exposed to the test substance at all exposure levels demonstrated statistically reduced food consumption over the entire exposure period (test day 0–91) when compared to the air exposed control group. Females exposed to 4000, 5000 and 7500 ppm demonstrated statistically reduced food consumption on test day 0–7, with the 7500 ppm group maintained statistical significance for the entire in-life period and the 3000, 4000 and 5000 ppm groups demonstrating statistical reductions starting again on test day 28–35 that was fairly constant until the end of the study. The only statistical alteration observed in food efficiency was a significant reduction in females exposed to 7500 ppm on test day 0–7. Since the reductions in food consumption were not associated with adverse changes in body weights, body weight gains or food efficiency, the statistical alterations observed in food consumption by female rats were considered non-adverse.

OPHTHALMOSCOPIC EXAMINATION: One male rat exposed to 4000 ppm and one female rat exposed to 5000 ppm demonstrated retinal degeneration on test days 81 and 80, respectively. Since the retinal degeneration occurred with very low incidence and did not demonstrate a concentration-response relationship, these finding were considered non-adverse and not related to test substance exposure.

HAEMATOLOGY: There were no adverse changes in group mean haematology parameters in male or female rats at test day 92. A statistically significant and concentration-related increase in absolute reticulocyte count (ARET) was present in male rats exposed to 5000 or 7500 ppm (32% to 34% above the control). These changes were likely test substance-related. However, biologically relevant increases in ARET normally occur in response to a decrease in red cell mass (haemoglobin [HGB], hematocrit [HCT], and red blood cell count [RBC]), but there were no associated effects on red cell mass parameters in these groups. In addition, there were no microscopic changes suggestive of an effect of blood cells (e.g., increased extramedullary haematopoiesis in spleen) in either sex. Therefore, the minimal increase in ARET in the 5000 and 7500 ppm male groups was not considered adverse . Absolute neutrophil count (ANEU) was statistically higher in male rats exposed to 3000 ppm as compared to control at test day 92. This change was considered unrelated to treatment (and non-adverse) because the change did not occur in a concentration-related pattern.

CLINICAL CHEMISTRY: There were no test substance-related changes in group mean clinical chemistry parameters in male or female rats at test day 92. Blood urea nitrogen (BUN) was minimally lower in male rats exposed to 7500 ppm (17% below the control). A statistically different change was not observed in females at any concentration level tested and no changes in BUN were observed in a previous 90-day inhalation study with at concentrations up to 10000 ppm. Biologically relevant changes in BUN generally occur as increases rather than decreases. Therefore, based on the minimal nature of the change, as well as the direction of change (decreased rather than increased), this lower BUN in the 7500 ppm male group was considered to be unrelated to treatment and non-adverse. Triglycerides (TRIG) was moderately decreased in male rats exposed to 7500 ppm (47% below the control). TRIG was also lower (22% below the control) in the 4000 and 5000 ppm males (not statistically significant). These decreases were likely secondary to decreases in body weight in these groups, as decreased serum TRIG are known to occur in rats following reduced food intake. There are no known adverse effects associated with decreases in TRIG. Therefore, the decrease in TRIG in these groups was likely a secondary effect of treatment, but was considered to be non-adverse. Glucose (GLU) was minimally decreased in male rats exposed to 5000 or 7500 ppm (10% and 16% below the control, respectively). These decreases in GLU values were also interpreted as secondary to decreases in body weight in these groups, as decreased serum GLU is known to occur in rats following reduced food intake. This change was likely a secondary effect of treatment, but was considered to be non-adverse because of its minimal nature. Similar changes in GLU were not present in females at any exposure concentration. Minimally higher total protein (TP) (6% to 10% above the control), characterized by minimally increased group means for both albumin (ALB) and globulin (GLOB) (7% to 12% above the control for ALB; 3% to 8% above the control for GLOB) was present in all treated male groups (variable statistical significance). These differences did not occur in a clear concentration-related manner across the concentrations tested. No statistically significant changes in any protein parameters were present at any exposure concentration in females. Therefore, these changes in protein parameters were likely unrelated to treatment. Regardless, based on their minimal nature, these differences in protein parameters were considered to be non-adverse.

URINALYSIS: There were no adverse changes in group mean urinalyses parameters in male or female rats. Urine volume (UVOL) was increased in male rats at all exposure levels (variable statistical significance). Increases in urine volume were accompanied by appropriate decreases in urine micro total protein (UMTP) concentration and in urinary specific gravity (SG) in males. These changes in UVOL, SG and UMTP were considered treatment related and represent the expected finding following exposure to a fluorine-containing compound. The diuretic response to fluorine-containing compound is characterized by increase in the UVOL, and associated decrease in SG. Although changes in these urinary parameters were related to treatment, they were considered to be non-adverse because there were no adverse changes in renal histopathology or clinical chemistry values (serum creatinine, BUN) suggestive of treatment related adverse renal functional or morphological effects. pH was minimally increased at all exposure levels in both sexes (variable statistical significance). Most individual values for pH were within the laboratory reference interval (males 6.0 – 8.0; females 6.0 – 7.5) and the remaining values were only slightly outside that interval (pH is reported in increments of 0.5). There were no other urinalyses, clinical chemistry or renal histopathology changes suggestive of kidney injury in either sex. Therefore, this minimal increase in urinary pH was considered possibly treatment related but non-adverse. Increased incidence of renal epithelial cells (both sexes) and finely and/or coarsely granular casts (males only) were observed microscopically in the sediment of exposed animals. The renal epithelial cells and casts were not associated with any adverse alteration in urinalysis parameters, clinical chemistry, or renal microscopic changes suggestive of kidney injury in either sex. Also, in a previous 90-day subchronic exposure study with the test substance at exposure levels up to 10000 ppm neither renal epithelial cells nor casts were observed in the urinalysis. Therefore, the increase in urinary granular cast/renal epithelial cells in test substance exposed groups was interpreted as potentially treatment related but non-adverse.

ORGAN WEIGHTS: There were no test substance-related effects on organ weights in this study. Mean absolute weights of brain and heart were lower in male rats exposed to 7500 ppm of test substance as compared to controls (statistically significant); and mean weights of brain, epididymides, kidney, liver, lungs, and testes relative to body weight were higher in male rats exposed to ≥3000 ppm of test substance as compared to controls (variable statistical significance). These changes occurred without correlative changes in other weight parameters for these organs and without correlative microscopic findings. These organ weight changes were likely the result of the slight decreases (variable statistical significance) in body weight in ≥3000 ppm males. Mean heart weight relative to body weight was increased 8% as compared to controls (statistically significant) in male rats exposed to 5000 ppm of the test substance. This change did not occur in a concentration-related manner and was not associated with changes in mean absolute and relative (% brain weight) heart weight. In addition, no test substance-related heart weight changes occurred in female rats in any concentration group. Therefore, the change in mean heart weight relative to body weight in 5000 ppm male rats was considered spurious and unrelated to treatment. In the 7500 ppm females, mean weight of ovaries relative to body weight was increased 22% as compared to controls (statistically significant). This change in ovarian weight occurred without correlative changes in other weight parameters for this organ and without correlative microscopic findings. Thus, the change in ovaries relative to body weight was also considered to be the result of decreased body weight (decreased 11% compared to controls; statistically significant) in this group.

HISTOPATHOLOGY: NON-NEOPLASTIC: Test substance-related, non-adverse, microscopic findings were observed in the teeth, nasal bones and femur/knee joints of rats exposed by inhalation to the test substance for approximately 90 days.

TEETH: Incomplete decalcification of enamel was observed in the distal region of the upper incisors (as seen in Level I of the nose) of male rats exposed to ≥ 3000 ppm of the test substance. Since the morphological features of this change were similar in all the affected animals, the change was not graded for severity but rather was recorded as ‘present’ when observed microscopically. Microscopically, the change was characterized by the presence of amorphous basophilic material within the normally empty enamel space. This material likely represents retention of incompletely mineralized enamel, which is normally washed out in the decalcification and tissue processing procedures. The enamel is a thick layer formed by the ameloblasts. In routine tissue processing, it is lost by decalcification leaving the enamel space. During exposure to some fluoride-containing compounds, fluoride can be retained in the enamel as fluoridated apatite or fluorapatite, which is mostly insoluble and can be resistant to acid decalcification. Since metabolism of this test substance may release free fluoride, the enamel changes are likely the result of fluorapatite formation which imparted resistance to the acid decalcification process used to process tissues in this study. There were no changes in ameloblasts–the enamel forming cells of the tooth–or in general dentition in affected animals. Therefore, the incomplete decalcification of enamel was considered non-adverse. In addition to incisors, incomplete decalcification of upper molars, nasal turbinates and palatine bone was also observed in the posterior nose (level III and IV of the nose) of few treated males and females.

FEMUR: Incomplete decalcification of bone trabeculae in the femur was present in all treated males and 7500 ppm females. Incomplete decalcification of bone trabeculae was mainly observed in the epiphyseal and metaphyseal areas of femur and tibia and was characterized by the presence of deep basophilia of the matrix of bone trabeculae consistent with retention of mineral. Osteoblasts and osteoclasts were morphologically normal. As noted for the enamel changes, this finding in the bone likely represents incomplete decalcification of bone during the tissue decalcification procedure due to acid resistance imparted by the formation of fluorapitite. Incomplete decalcification of bone trabeculae was not associated with any morphological changes in osteoblasts or osteoclasts and was considered non-adverse.

OTHER FINDINGS
- MICRONUCLEUS EVALUATION: See study DI.K1.90Day.InhVap.MicrNuc.R.D-17453-785-1.KD in Section 7.6.2 for details on Micronucleus test

Effect levels

1

Dose descriptor:

NOAEC

Effect level:

5 000 other: ppm (nominal) (33548 mg/m3)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: test substance-related reductions in body weights and food consumption at 7500 ppm

2

Dose descriptor:

NOAEC

Effect level:

7 500 other: ppm (nominal) (50322 mg/m3)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: highest exposure concentration tested

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
NOAEC in male rats = 5000 ppm (33548 mg/m3)
NOAEC in female rats = 7500 ppm (50322 mg/m3)

Executive summary:

Five groups of 10 male and 10 female Crl:CD(SD) rats each were exposed whole body, 6 hours per day, 5 days per week to 0, 3000, 4000, 5000 or 7500 ppm of the test substance over a 90 day period for a total of 65 exposures. Animals were weighed, observed for clinical signs of toxicity and had body weight and food consumption evaluations weekly. Animals had an ophthalmology examination prior to commencement of exposure and during week 12 of exposure. Five animals per sex per exposure concentration had blood collected following the fourth exposure, after approximately 4 weeks of exposure and at the time of final sacrifice for micronucleus evaluation. After approximately 90 days of exposure all animals were fasted, blood and urine samples collected and sacrificed one day postexposure for anatomical pathology evaluation, including microscopic tissue evaluation. 

Test substance-related adverse effects on body weight and food consumption were observed in male rats exposed to 7500 ppm. Male rats exposed to 7500 ppm began demonstrating statistically significant reductions in body weight and food consumption, when compared to the air-exposed control group, on test day 14 which lasted until the end of the study. At the end of the exposure period, the mean body weight of males exposed to 7500 ppm was 16% lower than the mean body weight of the control group. The reduced body weights in the 7500 ppm male exposure group correlated with statistically significant reductions in mean daily food consumption over the exposure period; no alterations in food efficiency were observed. No test substance-related changes in body weight, food consumption or food efficiency were observed in males exposed to <5000 ppm or in female rats exposed to any concentration over the course of this study. No test substance-related adverse clinical signs of toxicity or adverse changes in ophthalmology evaluation of animals were observed in this study. The test substance did not have an effect in the micronucleus assay in any animals exposed during this study as assessed by the number of micronucleated polychromatic erythrocytes. There were no adverse findings in the clinical pathology, organ weight, or gross pathological evaluations of male and female rats exposed to the test substance in this study. Test substance-related, but non-adverse microscopic findings, were observed in the teeth and femur/knee joints of all male and female rats exposed to the test substance. These changes included incomplete decalcification of enamel in the distal region of the upper incisors, no changes in ameloblasts or dentition were observed in the affected animals. Incomplete decalcification of bone trabeculae in the femur was also present in all males exposed but only in females exposed to 7500 ppm; osteoclasts and osteoblasts were morphologically normal. These microscopic findings are consistent with exposure to a fluorine-containing test substance and were not associated with any histopathological changes suggestive of tissue injury or adverse functional consequences in these tissues and, therefore, were not considered to be adverse. Under the conditions of this study, the no-observed-adverse-effect-concentration (NOAEC) for the test substance in male rats was 5000 ppm (33548 mg/m³) based upon test substance-related reductions in body weights and food consumption observed in animals exposed to 7500 ppm. The NOAEC for female rats was 7500 ppm (50322 mg/m³), the highest exposure concentration tested in this study.