Reaction mass of (3E)-1,1,1,2,2,3,4,5,6,6,7,7,7-tridecafluoro-5-methoxyhept-3-ene and (2E)-1,1,1,2,3,4,5,5,6,6,7,7,7-tridecafluoro-4-methoxyhept-2-ene and (3E)-1,1,1,2,2,4,5,5,6,6,7,7,7-tridecafluoro-3-methoxyhept-3-ene

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:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

Remarks:

Conducted according to the guideline in effect at the time of study conduct.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

Remarks:

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:

(2E)-1,1,1,2,3,4,5,5,6,6,7,7,7-tridecafluoro-4-methoxyhept-2-ene; (3E)-1,1,1,2,2,3,4,5,6,6,7,7,7-tridecafluoro-5-methoxyhept-3-ene; (3E)-1,1,1,2,2,4,5,5,6,6,7,7,7-tridecafluoro-3-methoxyhept-3-ene

EC Number:

700-755-2

Molecular formula:

C8H3F13O

IUPAC Name:

(2E)-1,1,1,2,3,4,5,5,6,6,7,7,7-tridecafluoro-4-methoxyhept-2-ene; (3E)-1,1,1,2,2,3,4,5,6,6,7,7,7-tridecafluoro-5-methoxyhept-3-ene; (3E)-1,1,1,2,2,4,5,5,6,6,7,7,7-tridecafluoro-3-methoxyhept-3-ene

Details on test material:

- Purity: 99.87%

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: approximately 55 days old
- Weight at study initiation: Males - weighed between 238.1 and 284.1 g; Females - weighed between 172.1 and 206.9 g
- Fasting period before study: Except during exposure, food and tap water were available ad libitum. During the urine collection period, rats were fasted overnight for at least 15 hours; water, however, was available ad libitum
- Housing: animals were housed 2-3 per cage in suspended, solid-bottom caging with Nestlets as enrichment.
- Diet: PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002, ad libitum
- Water: tap water, ad libitum
- Acclimation period: quarantined for 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26ºC
- Humidity (%): 30-70%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: nitrogen and air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
The chamber atmospheres were generated by flash evaporation of the liquid test substance in nitrogen. The test substance liquid was metered to a heated 1000 mL 3-neck generation flask using either a Cole-Parmer Masterflex pump and a Fluid Metering Inc. (FMI) piston or a Harvard Apparatus model 22 Infusion pump (or syringe drive). The flask was heated to 250°C using an Electrothermal Unimantle which was controlled by the Camile Inhalation Toxicology Automated Data System (CITADS). The generation nitrogen supply was metered to the generation flask by Brooks model(s) 5850E (or 5850I or 5851E or 5851I) mass flow controllers. The test item vapour and nitrogen mixture left the flask and entered a transfer tube where supplemental chamber air was added to the mixture via Brooks models 5850E (or 5850I or 5851E or 5851I) mass flow controllers. The gas mixture entered the chamber through a tangential turret at the top of the chamber. The test atmosphere was exhausted from the bottom of the chamber through a dry-ice cold trap followed by a MSA charcoal/HEPA filter cartridge prior to discharge into the fume hood.

TEST ATMOSPHERE
- Prior to the start of the exposure phase, the distribution of the test substance was determined in the high-concentration chamber. Air samples were collected from 8 separate locations in the chamber and also in the middle of the chamber. Samples from the 8 separate locations were compared to the overall average and the test substance for determination of homogenously distributed test substance in the exposure chamber.
During the exposure, the vapour concentration of the test substance was determined by gas chromatography at least once per hour in the test chambers. Volumes of chamber atmosphere were continually drawn from the breathing zone of the animals and were directly injected into an Agilent model 6890 Network gas chromatograph (GC) equipped with an automated gas sample valve and a flame-ionization detector.

VEHICLE (if applicable)
- nitrogen and air

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

All samples were chromatographed isothermally at 150°C on a Model J&W DB-5 fused silica glass column. The atmospheric concentration of the test substance vapour was determined from a standard curve derived from gas standards. Standards were prepared prior to the exposure by injecting known volumes of the liquid test substance into Tedlar® bags containing known volumes of air.

Duration of treatment / exposure:

28 days

Frequency of treatment:

6 hours/day over a 4-week period (weekends and holidays excluded) for a total of 21 exposures

Doses / concentrations

Remarks:

Doses / Concentrations:
1000, 2500, 5000 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

20 animals/sex/dose (10 animals/sex/dose were used in an evaluation and recovery group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: During the pilot study, lethality was observed after repeated exposure to 7500 and 10000 ppm of the test item. Therefore, the highest targeted test substance concentration of 5000 ppm was selected for this 4-week inhalation toxicity study.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cage-site examinations to detect moribund or dead animals and abnormal behaviour and appearance among rats were conducted at the time of loading the animals into the chamber on exposure days, and at least once daily on non-exposure days.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed before the exposure period, twice a week during the exposure period, and at least once a week during the recovery period.

FOOD CONSUMPTION: 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

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The baseline examination was performed on all rats received for the study, prior to assignment to groups. Any rats with preexisting ophthalmology abnormalities were eliminated from consideration for use in the study. All surviving rats were examined prior to the 4-week sacrifice.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Yes, following the fourth exposure, following 4-weeks of exposure and following a 14-day recovery period
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: following the fourth exposure, following 4-weeks of exposure and following a 14-day recovery period
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: following the fourth exposure, following 4-weeks of exposure and following a 14-day recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.2] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Neurobehavioral evaluation, consisting of abbreviated functional observational battery (FOB) assessments and motor activity (MA), was conducted on all animals designated for neurobehavioral evaluation and recovery during acclimation (baseline) and on all surviving animals in that subset prior to the end of the exposure period.

OTHER: The Micronucleus Evaluation was conducted by flow cytometry, following published guidance. Peripheral blood samples were collected by tail vein from 5 animals per sex per group. Blood was collected from the first 5 animals numerically from rats designated for subchronic toxicity (cohort I). There were 2 blood collections, one after the third exposure and one following the animals’ final exposure. Blood collections occurred within 4 hours after the exposures. See Sec. 7.6.2 for DI.K1.InhVap.MicroNuc.R.18157-782.KD for additional details.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table No. 3)

All animals found dead, prior to or at scheduled sacrifice, underwent a gross evaluation. The necropsy included gross examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. Relative organ weights (percent of terminal body weight; percent of brain weight) for weighed organs were calculated. Terminal body weights determined just prior to necropsy were used in the assessment of organ weight changes. Organs from animals found dead, euthanized prior to scheduled sacrifice, or accidentally killed were weighed.

HISTOPATHOLOGY: Yes (see table No. 3)

All animals found dead, prior to or at scheduled sacrifice, underwent a gross and microscopic evaluation. The eyes, optic nerves, testes, and epididymides were preserved in Modified Davidson’s solution. All other tissues were preserved in 10% neutral buffered formalin. All tissues collected from animals designated for subchronic toxicity in the high-concentration and control groups, and from the animal that was found dead (tissue integrity permitting), prior to scheduled sacrifice were further processed to slides, stained with hematoxylin and eosin, and examined microscopically. Target organs (liver, larynx, and nose level 1) from animals in the low- and intermediate-concentration groups were evaluated microscopically. The remaining tissues collected from animals in the low- and intermediate-concentration groups designated for subchronic toxicity were evaluated microscopically to establish a no-observed-adverse-effect concentration (NOAEC) for potential test substance-related abnormalities. Tissues from animals removed from study (for other than out-of-range body weight on test day 1), were not processed for microscopic evaluation. Target organs from animals designated for neurobehavioral evaluation and recovery were evaluated microscopically to determine the reversibility or persistence of toxic effects observed at the end of the exposure period.

Statistics:

See Table 4. Significance was judged at p < 0.05. Separate analyses were performed on the data collected for each sex. For each parameter analyzed with a trend test, the test was applied to the data sequentially. If a significant treatment-response was detected, data from the top treatment group were excluded and the test repeated until no significant trend was detected.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional 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:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Sporadic incidences of superficial wounds and/or hair loss were observed in male animals exposed to 0 or 1000 ppm of the test item. These clinical observations were sporadic in nature, did not demonstrate a concentration-response relationship and were therefore not considered test substance-related or adverse. One male rat exposed to 1000 ppm was found to have slow breathing, decreased muscle tone and in moribund state on test day 27, and was euthanized. Histopathological evaluation of this animal indicated inflammation in the kidneys and urinary bladder with bacterial colonies in both kidneys, and the cause of death was attributed to urogenital tract inflammation.

BODY WEIGHT AND WEIGHT GAIN
Male rats exposed to 5100 ppm demonstrated a statistically significant reduction (5.7%) in body weight on the day of sacrifice (test day 30) when compared to the air-exposed control group. There were no changes in body weight values of female rats exposed to the test item. Male and female rats exposed to 5100 ppm demonstrated a statistically significant reduction in body weight gain from test days 8 to 11 and test days 11 to 15, respectively. These reductions were sporadic in nature, not associated with changes in body weight and are therefore considered non-adverse.

FOOD CONSUMPTION
Male rats exposed to 5100 ppm demonstrated a statistically significant reduction in food consumption from test days 1 to 8 and statistically significant increase in food consumption from test days 36 to 43 and males exposed to 1000 ppm demonstrated statistically elevated food consumption over the entire in-life phase of the study (test days 1 to 43). These statistical differences were considered sporadic in nature, not associated with adverse changes in body weight and therefore, non-adverse.

Female rats exposed to 5100 ppm demonstrated statistically significant reductions in food consumption on test days 1 to 8, 22 to 29 and 1 to 20. While statistically significant, these reductions were minimal in magnitude (<5.6%) when compared to the control group, were not associated with changes in body weight and were therefore, considered non-adverse.

OPHTHALMOSCOPIC EXAMINATION
One male control animal (0 ppm) demonstrated multifocal retinal changes following the 4-week exposure period. There were no adverse findings observed at any exposure level of the test item.

HAEMATOLOGY
Red cell mass parameters (haemoglobin (HGB), red blood cells (RBC), and haematocrit (HCT)) were minimally decreased (≤ 6% for all parameters) in the 2500 and 5100 ppm males and 5100 ppm female groups (variable statistical significance). The changes in red cell mass parameters were associated with statistically significant increases (25-38% above controls) in reticulocytes (ARET) and consequent changes in red cell indices: decreased mean corpuscular haemoglobin/haemoglobin concentration, increased mean corpuscular volume, and increased red cell distribution width (differences were all generally <7% compared to control with variable statistical significance). Taken together, the changes in ARET and red cell indices are consistent with increased erythropoiesis in response to the minimal decrements in red cell mass. An increase in platelet counts in the 2500 and 5100 ppm male groups (9 and 14%, respectively, above controls) was associated with increased erythropoiesis. Based on the pattern and consistency of changes in these haematology parameters, they were considered to be test substance-related. However, based on their minimal nature, they were considered non-adverse. There were no statistically significant changes in red cell mass parameters following the 14-day recovery period. Variable statistically significant changes in ARET and some red cell indices were present in the 2500 and 5100 ppm male and 5100 ppm female recovery groups, but absent correlative changes in red cell mass parameters, these changes were considered non-adverse.

There were no other test substance-related or adverse haematology findings. The following statistically significant changes in mean haematology parameters were considered to be unrelated to exposure and non-adverse.
* Higher total white cell count (WBC) due to higher absolute lymphocyte count (ALYM) was present in the 5100 ppm male group. WBC and ALYM were increased to 26 and 27% above control, respectively. Similar increases (30 and 32% above control, respectively) were present in this group following the two-week recovery period. Statistical significance in this group was primarily due to fewer animals with low WBC and ALYM (compared to control), as individual values for most animals in this group were within the range of study controls. In addition, there were no correlative exposure-related inflammatory changes noted microscopically and no changes in white cell parameters in female rats at any of the concentrations tested. Therefore, the white cell changes in the 5100 ppm male group were considered to be unrelated to exposure.
* Higher absolute basophil count (ABAS) in recovery group males exposed to 2500 ppm was considered to be unrelated to exposure, as it did not occur in an exposure-related manner.
* Higher absolute large unstained cells (ALUC) in males exposed to 1000 or 5100 ppm was considered to be unrelated to exposure, as similar findings were not observed in the 2500 ppm group.

Coagulation:
There were no test-substance-related changes in coagulation parameters at any of the exposure groups tested. Activated partial thromboplastin time (APTT) was longer in females exposed to 2500 ppm. This statistically significant change was considered to be unrelated to exposure and non-adverse because it did not occur in an exposure-related manner.

CLINICAL CHEMISTRY
Serum proteins:
Statistically significant increases in albumin (ALB) and globulin (GLOB), with resulting increases in serum total protein (TP), were present in males at all exposure concentrations. TP was increased 6, 12, and 15% above control in the 1000, 2500, and 5100 ppm groups, respectively. There were no statistically significant changes in serum proteins in female rats at any of the exposure concentrations tested. Increases in both ALB and GLOB are most commonly associated with dehydration. Clinically relevant dehydration is associated with decrements in body weight and weight gain, and minimal decrements in body weight parameters (approximately 6% less than control) were observed only in the 5100 ppm male group. Therefore, in the 5100 ppm male group, the presence of increased serum proteins in association with decreased body weight was considered to be indicative of an adverse, albeit minimal, effect on hydration status (dehydration), likely occurring secondary to diuresis. However, there were no exposure-related changes in body weights at the lower exposure concentrations, indicating physiological maintenance of hydration status at these concentrations. Therefore, the changes in serum protein in these groups were considered to be exposure-related but non-adverse.

There were no statistically significant changes in ALB or GLOB in the 2500 or 5100 ppm recovery groups. TP was statistically, but minimally (≤5%), higher in these groups. However, total protein values for individual animals in these groups were all within laboratory reference intervals (5.6-6.8 g/dL), and statistical significance was primarily the result of a relatively low mean TP in the male recovery group controls (5.9 g/dL versus historical mean of 6.2 g/dL). Therefore, the statistically higher TP in the 2500 and 5100 ppm male groups was considered to be a spurious finding.

Other:
All other statistically significant clinical chemistry and urinalysis findings were considered to be unrelated to exposure to the test material or non-adverse.

Alanine aminotransferase (ALT) was higher (26% above the control) in males exposed to 5100 ppm, and sorbitol dehydrogenase (SDH) was higher in males exposed to 2500 or 5100 ppm (40% and 49% above the control, respectively). These minimal changes (less than 2-fold higher than control in all cases) were not associated with statistically significant changes in other liver-related parameters (alkaline phosphatase, aspartate aminotransferase) and microscopic liver changes were limited to hepatocellular hypertrophy without evidence of liver cell injury. Therefore, the minimal changes in liver enzymes were considered to be of uncertain relationship to exposure but non-adverse.

Bilirubin (BILI) was higher (20% above the control) in males exposed to 5100 ppm at the end of the exposure period. However, all individual values for BILI in this group were within the laboratory reference interval (males 0.08-0.18 mg/dL) for this parameter. In addition, there were no statistically significant changes in BILI in the 5100 ppm males following the 14-day recovery period. Therefore, this change was considered to be spurious and non-adverse.

Cholesterol (CHOL) was minimally increased in males and females exposed to 5100 ppm (27% and 33% above the control, respectively). CHOL was similarly elevated in the 5100 ppm males and females following the 14-day recovery period (51% and 26% above the control, for males and females, respectively). CHOL changes were influenced by one male (animal 415) and one female (animal 467) whose higher cholesterol values were observed during exposure and recovery. Most other individual CHOL values were within the laboratory reference interval (males 31-90 mg/dL; 46-115 mg/dL females) for this parameter. In addition, triglycerides (TRIG) were minimally increased in females in the 2500 and 5100 ppm exposure groups (37% and 51% above the control, respectively). Such minimal changes in CHOL and TRIG are frequent findings in toxicology studies and are considered to represent minor alterations in lipid metabolism that do not adversely affect the animal’s health. As such, these statistically significant changes in serum lipids are considered to be non-adverse. A statistically higher TRIG in the 2500 ppm female recovery group did not occur in an exposure-related manner and was considered to be unrelated to exposure to the test substance.

Calcium (CALC) was increased in males exposed to 2500 or 5100 ppm. These changes were minimal (3% and 5% above the control, respectively) and represent appropriate physiological increases in calcium due to the increases in albumin seen at these exposure concentrations. Forty percent of serum calcium is bound to albumin. As such, these changes in calcium were considered to be non-adverse. The changes in serum calcium were reversible following the 14-day recovery period. Statistically higher serum calcium was present in the 2500 ppm male recovery group but this change was minimal (2% above controls) and did not occur in an exposure-related manner and was, therefore, considered to be unrelated to exposure to the test substance.

Inorganic phosphorus (IPHS) was increased in males exposed to 1000, 2500, or 5100 ppm at the end of the exposure period. These increases were considered to be test substance related, and increased IPHS is known to occur following exposure to fluorinated compounds. However, these changes were minimal (7%, 7%, and 10% above the control, respectively) and values in all individual animals (except one animal in the 5100 ppm group) were within laboratory reference intervals (6.4-8.7 mg/dL). In addition, the changes in IPHS were reversible, as there were no statistically significant changes in IPHS in any exposed group following the 14-day recovery period. Therefore, these changes in serum phosphorus were considered to be non-adverse.

Chloride (CL) was lower in the 5100 male group. This decrease was minimal (2% lower than controls) and all individual CL values in this group were within laboratory reference intervals. In addition, there were no statistically significant differences in CL in any exposed group following the two-week recovery period. Therefore, the minimally lower CL in the 5100 ppm male group was considered to be non-adverse and likely spurious.

Blood Urea Nitrogen (BUN) and Creatinine (CREA) were lower (16% below the control for both values) in the 5100 ppm female group. There were no statistically significant changes in these parameters in any exposure group following the 14-day recovery period. These changes may have been test substance-related but based on the direction of change, decreased rather than increased, these changes were not considered to be adverse. CREA was also lower in the 1000 and 2500 ppm female groups. However, the differences were minimal (< 10% below control) and these differences did not occur in a clear exposure-related manner. Therefore these changes were likely spurious but regardless, based on the direction of change, these changes were also considered to be non-adverse.

The following statistically significant changes in mean clinical chemistry parameters were considered to be unrelated to exposure and non-adverse because they did not occur in an exposure-related manner or occurred only after approximately 2 weeks of recovery: Aspartate aminotransferase (AST) was lower in males exposed to 2500 ppm and in recovery group females exposed to 5100 ppm. Bilirubin (BILI) was lower in recovery group males exposed to 2500 ppm. Glucose (GLUC) was higher in recovery group males exposed to 1000 ppm and recovery group females exposed to 2500 or 5100 ppm.

URINALYSIS
Plasma and Urine Fluoride: Plasma fluoride was increased in the 2500 and 5100 ppm male and female groups, and urine fluoride was increased in all male and female exposure groups. The test material is a mixture composed primarily of perfluorinated heptene ethers, and the increases in plasma and urine fluoride indicate that metabolism of the parent material includes liberation of free fluoride. Following the 14-day recovery period, plasma fluoride concentration was the same as controls in all male and female exposure groups. Urine fluoride levels had not returned to control levels following the 14-day recovery but were decreased to 48, 24, and 15% of their previous (end of the exposure) values in the 1000, 2500, and 5100 ppm male groups, respectively. In females, urine fluoride levels were decreased to 42, 23, and 10% of the end-of-exposure values, respectively.

Urine Volume and Specific Gravity: Urine volume was increased and urine specific gravity was decreased in males at all exposure concentrations and in females at 2500 and 5100 ppm. Increased urine volume and decreased urine specific gravity are consistent with diuresis. In the 2500 or 5100 ppm female groups, the changes in urine volume and urine specific gravity were statistically significant. In males, these urinalysis changes were not statistically significant. However, individual values for urine volume and specific gravity in several individual male rats in all exposure groups were above and below, respectively, the range of study control values. Diuresis is known to occur in association with renal elimination of free fluoride, and as noted above, plasma fluoride was increased in the 2500 and 5100 ppm male and female groups, and urine fluoride was increased in all exposed male and female groups. Therefore, the changes in urine volume and specific gravity in test substance-exposed groups were considered to be test substance-related and the result of metabolism of the parent material to free fluoride and subsequent urinary elimination of fluoride. In the 5100 ppm male group, diuresis was associated with an increase in serum proteins and decrease in body weight indicative of dehydration (see discussion below). Therefore, the urinalysis changes in males at 5100 ppm were considered to be adverse. Urine volume and specific gravity in all male and female exposure groups were similar to controls following the 14-day recovery period. In males, statistically significant differences in urine volume and specific gravity were observed in the 2500 ppm exposure group. These changes did not occur in an exposure-related manner and were therefore considered to be unrelated to exposure to the test substance.

There were no other test substance-related or adverse urinalysis findings. The following statistically significant changes in mean urinalysis parameters were considered to be unrelated to exposure and non-adverse. Higher pH in females exposed to 1000 or 51000 ppm was considered to be unrelated to exposure, as similar findings were not observed in the 2500 ppm female group or in any male exposure group.

ORGAN WEIGHTS
A treatment-related increase in liver weights occurred at 1000, 2500, and 5100 ppm in males and at 5100 ppm in females. In males mean liver relative to body weight was increased 16.9%, 36.3%, and 62.7% at 1000, 2500, and 5100 ppm, respectively. In females mean liver relative to body weight was increased 36.3% at 5100 ppm. At the recovery necropsy liver weights relative to body weight were still increased 12.0% and 25.7% at 2500 and 5100 ppm, respectively in males and 16.7% at 5100 ppm in females. Also in females at the recovery necropsy an increase in mean liver relative to body weight of 8.0% at 2500 ppm was statistically significant, however this small difference was not likely biologically relevant. Other weight differences in the 5100 ppm male were considered secondary to the 5.7% bodyweight decrement in this group compared to the controls while differences in other groups at either the terminal or recovery necropsy were considered spurious and not related to compound administration.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related microscopic findings were noted in the liver, larynx, and level 1 of the nose. In the liver panlobular hepatocellular hypertrophy was noted in both sexes at 5100 ppm while centrilobular hepatocellular hypertrophy was noted in males at 1000 and 2500 ppm. At the recovery necropsy centrilobular hepatocellular hypertrophy was present in the 5100 ppm males. Affected hepatocytes were enlarged with increased homogenous, eosinophilic cytoplasm. Hepatocellular hypertrophy is generally considered an adaptive response associated with metabolism of administration of xenobiotics and is not considered adverse.

In the larynx, hyperplasia/squamous metaplasia of respiratory epithelium of minimal severity was present in all concentration groups. At the recovery necropsy effects were still present in the 1000 and 5100 ppm group males and in a single 5100 ppm group female. Changes were characterized by increased thickness of the covering epithelium and some flattening of the lining epithelium characteristic of squamous cells. This change is considered an adaptive response secondary to laryngeal irritation.

In level 1 of the nose (the most anterior section) hypertrophy of goblet cells was increased in all concentration groups at both the terminal and recovery necropsies. Goblet cells of the nasal septum were increased in size and goblet cells extended up and beyond the septum. As with the change in the larynx, this change is considered an adaptive response secondary to nasal irritation. Also in the level 1 nose section, test substance-related changes were noted in the incisor teeth consistent with fluoride exposure in males at 2500 and 5100 ppm. These included degeneration/atrophy of ameloblastic epithelium, lamination of dentin, and incomplete decalcification of enamel and/or dentin. Degeneration/atrophy of ameloblastic epithelium of moderate severity was noted in a single recovery male. The change was characterized by disorganization and attenuation of ameloblastic epithelium in the incisor cross section at level 1 of the nose and was considered adverse.

Lamination of dentin was noted in 7/10 males at 5100 ppm at the terminal necropsy. At the recovery necropsy, lamination of dentin was noted in 2/10 males at 5100 ppm and 1/10 males at 2500 ppm. Lamination of dentin was characterized by the presence of concentric (about the pulp cavity) basophilic rings within the dentin that likely represents fluoride-induced changes in mineralization of dentin. The lamination in these animals was very minimal and the finding was not considered adverse. Finally, incomplete decalcification of enamel/dentin was noted in 2/10 males at 5100 ppm at the terminal necropsy, 5/10 males at 5100 ppm at the recovery necropsy, and 4/10 males at 2500 ppm at the recovery necropsy. This change was limited to retention of mineralized material in the enamel space of the incisor at level 1 of the nose. Incomplete decalcification of teeth are likely the result of fluoride-associated resistance to the acid decalcification procedure used in this study, as fluoride can reduce the acid solubility of mineral in teeth and bones. As such, this finding was considered to be indicative of exposure to a metabolized fluoride, but non-adverse.

Other microscopic findings in these animals were typical of those typically seen in rats of this strain and age and were considered incidental and not related to compound administration.

OTHER:
- Micronucleus Evaluation; See Sec. 7.6.2 for DI.K1.InhVap.MicroNuc.R.18157-782.KD for additional details.

Effect levels

1

Dose descriptor:

NOAEC

Effect level:

2 500 other: ppm (37025 mg/m3)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Test substance-related diuresis-induced dehydration associated with increased serum total protein, albumin and globulins as well as a single incidence of degeneration/atrophy of ameloblastic epithelium observed in males exposed to 5100 ppm.

2

Dose descriptor:

NOAEC

Effect level:

5 100 other: ppm (75531 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

Any other information on results incl. tables

Table 1. Serum values for Male Rats

Male Rat	Day(s) Relative to Start Date		0 ppm	1000 ppm	2500 ppm	5000 ppm
Albumin g/dL	30	Mean	3.4	3.5	3.8	3.8
		% Diff	-	5.1	11.9	14.6
	47	Mean	3.4	3.4	3.5	3.5
		% Diff	-	1.2	3.6	3.0
Globulin g/dL	30	Mean	2.8	3.0	3.1	3.3
		% Diff	-	6.6	11.2	16.1
	47	Mean	2.6	2.6	2.7	2.8
		% Diff	-	3.1	5.5	8.6
Total Protein g/dL	30	Mean	6.2	6.5	6.9	7.1
		% Diff	-	5.8	11.6	15.3
	47	Mean	5.9	6.0	6.2	6.2
		% Diff	-	2.0	4.4	5.4

Table 2. Urine values for Male Rats

Male Rat	Day(s) Relative to Start Date		0 ppm	1000 ppm	2500 ppm	5000 ppm
Urine volume mL	30	Mean	11.6	16.9	22.4	17.9
		% Diff	-	45.6	92.9	54.1
	47	Mean	19.3	28.7	36.2	20.0
		% Diff	-	48.6	87.6	3.6
Specific Gravity	30	Mean	1.031	1.028	1.029	1.027
		% Diff	-	-.03	-0.2	-0.4
	47	Mean	1.027	1.019	1.013	1.023
		% Diff	-	-0.8	-1.3	-0.4
Total Protein mg/dL	30	Mean	134	140	197	206
		% Diff	-	4.9	47.0	54.1
	47	Mean	108	77	67	119
		% Diff	-	-28.8	-38.6	9.5

Applicant's summary and conclusion

Conclusions:

This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
Male NOAEL = 2500 ppm
Female NOAEL = 5100ppm

Executive summary:

The objective of this study was to assess the potential subchronic toxicity of the test item in rats as well as evaluate the ability of the test substance to induce micronuclei in bone marrow by analysing peripheral blood reticulocytes. Four groups of 20 male and 20 female Crl:CD(SD) rats each were exposed whole body, 6 hours per day, 5 days per week to 0.00 ± 0.00 (air-exposed control), 1000, 2500 or 5100 ppm (mean) over a 4-week period for a total of 21 exposures. Animals were weighed, observed for clinical signs of toxicity and had body weight and food consumption evaluations throughout the study. Following 4-weeks of exposure to the test item, groups of 10 male and 10 female rats from each exposure level were fasted, blood and urine samples collected for clinical pathology, and sacrificed one day following their final exposure for anatomical pathology evaluation including microscopic tissue evaluation. Five animals per sex per exposure concentration had blood collected following the fourth exposure and after approximately 4 weeks of exposure at the time of final sacrifice for micronucleus evaluation. Following a 14-day recovery period, groups of 10 male and 10 female rats were fasted, blood and urine samples collected for clinical pathology, and sacrificed for anatomical pathology evaluation including microscopic tissue evaluation.

 

Male rats exposed to 5100 ppm demonstrated a statistically significant reduction (5.7%) in body weight one day following their final exposure when compared to the air-exposed control group. There were no test substance-related adverse effects on body weight gain, food consumption, clincial signs of toxicity or in the ophthalmology exams in male or females at any exposure level to the test item. There were also no test substance-related effects on any neurobehavioral parameter evaluated in male or females at any exposure level. The test item did not have an effect in the micronucleus assay in any animals exposed during this study as assessed by the number of micronucleated reticulocytes.

 

Consistent with exposure to a fluorinated compound, plasma fluoride was increased in the 2500 and 5100 ppm male and female groups, and urine fluoride was increased in all male and female exposure groups. Following the 14-day recovery period, plasma fluoride concentrations were essentially the same as controls in all male and female exposure groups. Urine fluoride levels had not returned to control levels following the 14-day recovery but were decreased to 48, 24, and 15% of their previous (end-of-exposure) values in the 1000, 2500, and 5100 ppm male groups, respectively. In recovery group females, urine fluoride levels were decreased to 42, 23, and 10% of the end-of-exposure values, respectively.

 

Exposure of male rats to 5100 ppm was associated with increased urine volume and decreased urine specific gravity consistent with diuresis, increased serum proteins (albumin, globulin and total proteins), and decreased body weight. Increases in serum proteins occurring in association with decrements in body weight in the 5100 ppm male group were considered to be indicative of an adverse, albeit minimal, effect on hydration status (dehydration), likely occurring secondary to fluoride diuresis. Although evidence of diuresis was also present in males at 1000 and 2500 ppm and in females at 2500 and 5100 ppm, and minimal increases in serum proteins were present in the 1000 and 2500 ppm male groups, these changes were not associated with effects on body weight parameters, indicating that hydration status was not significantly altered at these exposure concentrations. Therefore, these changes were likely test substance-related but were considered to be non-adverse. Changes in urinalysis and serum protein parameters were reversible following the two-week recovery period. There were no changes in clinical pathology parameters considered to be adverse in males at 1000 and 2500 ppm or in females at any of the exposure concentrations tested.

 

Liver hepatocellular hypertrophy occurred in both sexes at 5100 ppm and in males at 1000 and 2500 ppm at the terminal necropsy and in males at 5100 ppm at the recovery necropsy. This is considered an adaptive response associated with metabolism of xenobiotics and non-adverse. Hyperplasia/squamous metaplasia of the larynx occurred in all exposure groups at the terminal necropsy and at 1000 and 5100 ppm in males at the recovery necropsy and in a single female at 5100 ppm at the recovery necropsy. Goblet cell hypertrophy/hyperplasia occurred in all exposure groups at both the terminal and recovery necropsy. These laryngeal and nasal changes are considered a non-adverse, adaptive response to irritation and would be expected to be recoverable over time. Microscopic evidence of fluoride exposure was noted in the incisor teeth of males at 2500 and 5100 ppm with degeneration/atrophy of ameloblastic epithelium noted in a single 5100 ppm male which was considered adverse. Other findings associated with the fluoride exposure included very slight lamination of dentin at 5100 and 2500 ppm (a single male affected at 2500 ppm), and incomplete decalcification of enamel/dentin at 2500 and 5100 ppm. These latter changes were not considered adverse as the lamination was very minimal and the incomplete decalcification, while indicative of exposure, is not an adverse finding in the animal.

 

 

Under the conditions of this study, the no-observed-adverse-effect-concentration (NOAEC) for the test item in male rats was 2500 ppm based upon test substance-related diuresis-induced dehydration associated with increased serum total protein, albumin and globulins as well as a single incidence of degeneration/atrophy of ameloblastic epithelium observed in males exposed to 5100 ppm. The NOAEC for female rats was 5100 ppm, the highest exposure concentration tested in this study. Under the conditions of this study, the test item did not induce biologically relevant increases in micronucleated RETs in rat peripheral blood