Registration Dossier

Administrative data

Description of key information

The NOAEL in a 90-day oral study with 6:2 FTOH is 5 mg/kg/day, based on changes observed in haematology and clinical chemistry parameters, urinalysis parameters (females only), urine fluoride, and histopathological effects in the liver,  which corrected for molecular weight differences between the test and read across substances, would yield an equivalent test substance  NOAEL of 5.95 mg/kg/day. 
The LOAEC in a 28-day inhalation study with 6:2 FTOH is 100 ppm (1490 mg/m3), based on low incidence of combined increases in serum ALT and bilirubin levels, and increased liver weights in females exposed to 100 ppm, , which corrected for molecular weight differences between the test and read across substances, would yield an equivalent test substance NOAEC of 1773 mg/m3.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
5.95 mg/kg bw/day
Study duration:
subchronic
Species:
mouse

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
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 study is used for read-across and therefore has been assigned a reliability of 2 (reliable with restrictions). Otherwise the study has a reliability of 1 (reliable without restriction).
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
yes
Remarks:
Serum gamma glutamyl transpeptidase and ornithine decarboxylase were not evaluated.
Qualifier:
according to
Guideline:
other: EEC Methods for the Determination of Toxicity Method B.8 Directive 92/69/EEC
Deviations:
no
Remarks:
Serum gamma glutamyl transpeptidase was not evaluated.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: between 235 and 300 grams (males); between 174 and 217 grams (females)
- Fasting period before study: no
- Housing: solid bottom caging with Sheperd’s(TM) Cob + PLUS(TM) as bedding and enrichment.
- Diet (e.g. ad libitum): ad libitum except during exposure and fasting periods prior to necropsy
- Water (e.g. ad libitum): ad libitum except during exposure
- Acclimation period: at least 6 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23° C
- Humidity (%): 43 to 70%
- Air changes (per hr): 6 to 10
- Photoperiod (hrs dark / hrs light): 12-hours dark / 12-hours light
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel and glass (NYU style) with a nominal internal volume of 750 L (test chambers) or 1000 L (control chamber). A tangential feed inside the chamber 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 individually placed in stainless steel, wire-mesh cages and exposed, whole-body, inside the exposure chamber.
- Source and rate of air: Houseline
- Method of conditioning air: Not reported
- System of generating test atmosphere: Chamber atmospheres for the low level chamber were generated by vaporisation of the test substance in air by passing air over the test substance contained in an impinger. Chamber atmospheres for the intermediate- and high-level chambers were generated by flash evaporation of the test substance in air with a heated, glass mixing flask. The test substance was metered into the mixing flask with a syringe infusion pump. The mixing flask was heated to approximately 175-180°C to vaporise the test substance. Houseline generation air, metered to the mixing flask by a mass flow controller, carried the vaporised test substance into a filtered and conditioned dilution air supply leading to the exposure chamber. The filtered and conditioned dilution air supply was monitored with a thermoanemometer. Heat tape was used to heat the glass connecting tube from the heated flask to the top of the chamber to ensure the test substance remained in the vapour state. Chamber atmospheres for the control chamber were generated similarly to the intermediate- and high-levels chambers except without the infusion pump or heat tape on the glass connecting tube from the heated flask to the chamber. The heating mixing flask, heat tapes, and dilution air were controlled and monitored by the Inhalation Toxicology Automated Data System. Chamber concentrations of test substance were controlled by varying the test substance feed rate to the heated flask, varying the amount of generation air, and/or varying the amount of dilution air.
- Temperature, humidity, pressure in air chamber: Temperature ranged from 17 to 23°C (targeted at 20-24°C). Relative humidity ranged from 43 to 70% (targeted at 30-70%). Oxygen concentration was 21%. Air pressure was not reported.
- Air flow rate: Not reported
- Air change rate: 6 to 10 air changes per hour
- Treatment of exhaust air: Exhausted through an exhaust manifold prior to discharge into the fume hood

TEST ATMOSPHERE
- Brief description of analytical method used: Atmospheric concentrations of the test substance were determined by gas chromatography at approximately 60-minute intervals during the daily exposures.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Known volumes of chamber atmosphere were drawn from the sampling port through a sampling train consisting of a midget, fritted, glass impinger containing acetone as the collection medium. Aliquots of impinger solution were injected into a gas chromatograph equipped with a flame ionisation detector. All samples were chromatographed isothermally at 95°C on a fused silica glass column with a 30-meter length, 530 μm inside diameter, and 3.0 μm film thickness. The atmospheric vapour concentration of the test substance was determined from a standard curve derived from liquid standards. Standards were prepared weekly by quantitative dilution of the liquid test substance in acetone. Upon completion of the exposures, sample results were transferred to the Inhalation Reporting and Analysis System, which collated sample calculations.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
23 exposures over a 4-week period (weekends excluded)
Remarks:
Doses / Concentrations:
0 ± 0, 1.0 ± 0.0084, 10 ± 0.057 or 100 ± 0.33 ppm (mean ± S.E.M.)
Basis:
analytical conc.
No. of animals per sex per dose:
20 males and 20 females in the control and 100 ppm dose groups; 10 males and 10 females in the 1 and 10 ppm dose groups.
The first 10 male and female rats per dose level were designated for sacrifice at the end of the exposure period. The remaining 10 male and female rats per dose level were designated for sacrifice at the end of the 1-month recovery period.
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: A previously performed acute inhalation study indicated low toxicity where the LC50 for the test substance was greater than 5.2 mg/L but less than 9.9 mg/L, where the 5.2 and 9.9 mg/L exposure atmospheres contained both vapour and aerosol phases of the test substance. Results from the acute and a one-week subchronic inhalation exposure with the test substance indicated that the maximum practically attainable vapour-only exposure atmosphere was approximately 100 ppm. Exposure of male and female rats to 100 ppm for 5 days resulted in minimal body weight losses that were approximately 6-8% less than the control animals and microscopic pathological findings that were consistent with exposure to a fluorine-containing test material. Therefore, the high concentration of the test substance for the 4-week study was selected to be 100 ppm with the intermediate and low concentrations being log orders of magnitude less than the high concentration.
- Rationale for animal assignment (if not random): Animals of each sex were selected for use on study from those released from quarantine based on adequate body weight gain and freedom from any ophthalmology abnormalities or clinical signs of disease or injury. They were distributed by computerised, stratified randomisation into study groups so that there were no statistically significant differences among group body weight means within a sex. The weight variation of selected rats did not exceed ± 20% of the mean weight for each sex. The first 10 rats in each group were designated for subchronic toxicity.
- Rationale for selecting satellite groups: The remaining rats in the control and high groups were designated for recovery.
- Post-exposure recovery period in satellite groups: At the conclusion of the 4-week exposure period, all animals designated for recovery were allowed to recover for approximately 4 weeks.
Positive control:
None.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Examinations to detect moribund or dead animals and abnormal behaviour and appearance among animals were conducted at the time of loading the animals into the chamber on exposure days, and at least once daily on non-exposure days.

GENERAL CLINICAL OBSERVATIONS: Yes
During the daily exposures, the response to an alerting stimulus was determined for the animals as a group within each exposure chamber. The alerting response was determined prior to the initiation of each exposure, 3 times during exposure, and after the conclusion of each exposure just prior to animal removal from the exposure chamber. Study technicians judged whether the group of animals within a given exposure chamber displayed a normal, diminished, enhanced, or absent alerting behaviour in response to a standardised auditory stimulus. In addition, study technicians observed the animals for clinical signs 3 times. Clinical signs observed were recorded for the animals collectively within a chamber, since individual animal identification was not visible to the observer. Immediately following each exposure, each rat was individually handled and examined for abnormal behaviour and appearance. Clinical signs of hair loss, and fur/skin stains associated with urination. During the recovery period clinical observations were continued.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At weighing, detailed clinical observations were conducted including (but not limited to) evaluation of fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, and unusual respiratory pattern), changes in gait, posture, response to handling, presence of clonic, tonic, stereotypical, or bizarre behaviour. Any abnormal clinical signs noted were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: At least once a week before exposure, and at least once a week during the recovery period.

FOOD CONSUMPTION: Yes
- 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: Yes
- From the food consumption and body weight data, the mean daily food efficiency was calculated.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
All surviving rats were examined prior to the 4-week sacrifice. Both eyes of each rat were examined by focal illumination and indirect ophthalmoscopy. The eyes were examined in subdued light after mydriasis had been produced.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On test day 31 for males and females at the end of the exposure period and at the end of the recovery period.
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: All animals.
- Parameters checked in Table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On test day 31 for males and females at the end of the exposure period and at the end of the recovery period.
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in Table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: On test day 31 for males and females at the end of the exposure period.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in Table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations and dose groups examined: All animals prior to test substance exposure and during the fourth week of exposure. Control and 100 ppm recovery group animals during the 8th week of the study.
- Battery of functions tested:
Abbreviated Functional Observation Battery: Sensory and motor function assessments were conducted by evaluating grip strength, responses to approach/touch, sharp auditory stimulus, and tail pinch, and pupillary constriction.
Motor Activity Evaluation: Rats were individually tested for 90 minutes in automated activity monitors.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see Table 4)
After approximately 28 days on study, 10 rats/sex/exposure concentration were sacrificed and necropsied for evaluation of subchronic toxicity. The rats were euthanized by isofluorane and exsanguination. Rats sacrificed by design were fasted on the afternoon before their scheduled sacrifice. A final sacrifice was performed on surviving rats following the final clinical pathology evaluation. The order of sacrifice was stratified across the groups with in a sex. Gross examination was performed on all rats and final body weights and organ weights were recorded. Of the 80 rats in the main study groups (10/sex/exposure level), 2 died before the 90-day terminal sacrifice. One group 1 male rat (108) and a group 3 female rat (358) were accidentally killed on Day 31 during blood collection and received a gross and microscopic examination. All other rats survived until scheduled termination.
After a post-exposure period of approximately 1 month, surviving rats from control and the high exposure level (100 ppm) were sacrificed and necropsied. Rats were euthanized, tissues collected, and organ weights recorded as described for the main study.

ORGAN WEIGHTS: Yes (See Table 4)
Mean absolute and mean relative organ weights (relative to final body weight; relative to brain weight) for weighed organs were calculated. Final body weights determined just prior to necropsy were used in the assessment of organ weight changes. Organs from rats accidentally killed were not weighed.

HISTOPATHOLOGY: Yes (see Table 4)
All tissues collected from rats designated for subchronic toxicity in the high-concentration and control groups, and tissues collected from early decedents were further processed to slides, stained with haematoxylin and eosin, and examined microscopically. Target organs from rats in the low- and intermediate-concentration groups were evaluated microscopically. The tibiofemoral joint (femur and tibia) and nose (teeth: sections from nose levels I and II contain the upper incisors) collected from rats in the low- and intermediate-concentration groups designated for subchronic toxicity were evaluated microscopically.
All 40 rats (20 males, 20 females) designated for the 1-month recovery period survived until the scheduled sacrifice. Potential target organs (incisor teeth and femur/knee joint) were examined microscopically from all recovery rats. As in the main study, examination of teeth in the recovery rats was limited to the 2 upper incisors present within the first 2 sections of the nose (levels I and II).
Statistics:
See Table 5.
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:
no effects observed
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:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
HAEMATOLOGY
There were no adverse changes in group mean haematology parameters in male or female rats at the end of the exposure period (test day 31). Minimal decreases in red blood cell mass parameters (red blood cell, haemoglobin, and haematocrit) in female rats exposed to 100 ppm were considered to be test substance-related, however, not adverse as decreases in red cell mass parameters of this magnitude (decreases in group means for red cell mass parameters were ≤ 5%) are not expected to affect red cell function-delivery of oxygen to tissues. After 1 month of recovery, group mean values for these measurements were similar to the respective control group and not statistically significant.

CLINICAL CHEMISTRY
Following 4 weeks of exposure to the test substance, concentration-related and adverse changes in clinical chemistry parameters were observed in male and female rats exposed to 100 ppm. In these animals, increased mean serum bilirubin levels were observed when compared to the control group and several females exposed to 100 ppm test substance exhibited increased alanine aminotransferase (ALT). Increased albumin (ALB) in male and female rats exposed to 100 ppm was considered to be test substance-related, however, not adverse. Following 4 weeks of recovery, group mean values for these clinical pathology parameters in both male and females were similar to the respective control group and not statistically different. The following statistically significant changes in group mean clinical chemistry parameters at test day 31 or after 1 month of recovery (test days 57/58) were considered to be unrelated to test substance exposure (and non-adverse) because the change did not occur in a concentration related pattern or occurred only after 1 month of recovery: cholesterol (CHOL) was minimally decreased in male rats exposed to 100 ppm after 1 month of recovery (25% below the control); triglycerides (TRIG) were minimally increased in male rats exposed to 1 or 10 ppm (30% and 37% above the control, respectively) at test day 31; total protein (TP) and globulin (GLOB) were minimally decreased in male rats exposed 10 ppm (3% and 5% below the control, respectively) at test day 31; and calcium (CALC) was minimally decreased in female rats exposed to 1 ppm (3% below the control) at test day 31.

URINALYSIS
There were no adverse changes in group mean urinalyses parameters in male or female rats following the exposure period (test day 31). The following statistically significant changes in mean urinalyses parameters were related to test substance, however, not adverse: Urine volume (UVOL) mildly increased in male rats exposed to 100 ppm (130% above the control). This increase in UVOL was accompanied by an appropriate decrease in urinary specific gravity (SG) and in urine total protein (UMTP) in male rats exposed to 100 ppm (2% and 46% below the control, respectively). These changes in UVOL, SG, and UMTP were considered to be related to treatment and represent the expected finding following exposure to a fluorine-containing compound. The diuretic response to a fluorine-containing compound is characterised by increase in the UVOL, and associated decrease in urinary specific gravity. Although changes in these urinary parameters were related to test substance exposure, they were considered to be non-adverse because there were no changes in renal histopathology or clinical chemistry values (Serum creatinine, BUN) suggestive of exposure related adverse renal functional or morphological effect. After 1 month of recovery, group means for UVOL, SG, and UMTP in males exposed to 100 ppm were similar to the respective control group and not statistically significant.

PLASMA AND URINE FLUORIDE
As expected following exposure to a fluoride-containing compound, plasma and urine fluoride were increased in some exposure groups. Statistically significant increases in plasma fluoride concentrations were observed at the end of exposure period (test day 31) in male and female rats exposed to 10 (females only) or 100 ppm. Following the one-month recovery (test day 57/58) group mean plasma fluoride values in male and female rats exposed to 100 ppm were lower, however, they remained statistically significantly increased compared to the control group.
Statistically significant increases in mean urine fluoride were present in male and female rats exposed to 1 (males only), 10 or 100 ppm at the end of the exposure period (test day 31). Following the one-month recovery (test day 57/58), mean urine fluoride was reduced; however, it remained statistically significantly increased in male and female rats exposed to 100 ppm.

NEUROBEHAVIOUR
Male rats exposed to 100 ppm test substance demonstrated decreased motor activity during the fourth week of exposure. The decreased motor activity in male rats exposed to 100 ppm had resolved following the 4-week recovery period. In the absence of other neurobehavioral changes in 100 ppm male rats or in female rats, the lower motor activity in 100 ppm males appears to be secondary to other systemic effects of the test substance, instead of a primary effect on the nervous system. No adverse effects on motor activity were observed in either males exposed to 1 or 10 ppm or female rats exposed to 1, 10, or 100 ppm.

ORGAN WEIGHTS
Male and female rats exposed to 100 ppm test substance demonstrated increased mean absolute liver weights as well as increased mean liver weights relative to body and brain weight one day following the last exposure. By the end of the recovery period, the liver weights were similar to control.
The following changes in group mean organ weight parameters were considered to be unrelated to test substance exposure and non-adverse:
At the end of the exposure period, mean relative (to body and brain weight) adrenal weight was higher in 100 ppm males; mean adrenal weight relative to body weight was also higher in the 10 ppm male group. These differences were statistically significant. However, these changes in adrenal weight parameters were not associated with changes in mean absolute adrenal weights, and no correlative microscopic findings were present in any animal in these groups. In addition, similar changes in adrenal weight were not present in any treated female group or in any sex following 1 month recovery. Therefore, increases in adrenal gland weight parameters were considered unrelated to test substance exposure.
Mean relative (to body weight) kidney weights and mean absolute and relative (to body weight and brain weight) uterine weights were higher compared to controls in the 100 ppm female recovery group. Both the kidney and uterine weight differences in the recovery animals were interpreted to be spurious since test substance-related kidney and uterine weight effects were not observed following the 28-day exposure period.
All other organ weight changes were considered incidental and unrelated to test substance administration.

HISTOPATHOLOGY
There were no test substance-related adverse microscopic findings at any of the exposure concentrations tested. Test substance-related, but non-adverse, microscopic findings were present in the teeth (incisors) and sections of long bones (femur and tibia) at the termination of exposure and following the 1-month recovery period. These changes consisted of increased lamination of dentin of the incisor teeth and incomplete decalcification of enamel of the incisors and the bone trabeculae in the tibia and femur. These findings were consistent with exposure to a fluorine-containing test substance and were not associated with any histopathological changes suggestive of tissue injury or any adverse functional consequences in these tissues. Therefore, these microscopic findings were not considered to be adverse.
Dose descriptor:
NOAEC
Effect level:
10 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based upon increased serum ALT and bilirubin levels (females) and increased liver weighs in both male and females exposed to 100 ppm.
Dose descriptor:
LOAEC
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based upon increased serum ALT and bilirubin levels (females) and increased liver weighs in both male and females exposed to 100 ppm.
Critical effects observed:
not specified
Conclusions:
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
The no-observed-adverse-effect conentration (NOAEC) was 10 ppm and the LOAEC was 100 ppm for male and female rats based upon increased serum ALT and bilirubin levels (females) and increased liver weighs in both male and females exposed to 100 ppm.
Executive summary:

Four groups male and female Crl:CD(SD) rats were exposed whole body 6 hours/day, 5 days/week to 0 ± 0, 1.0 ± 0.0084, 10 ± 0.057 or 100 ± 0.33 ppm test substance over a 4-week period for a total of 23 exposures. Ten male and 10 female rats exposed to 0 (control) or 100 ppm were then subject to an approximate 4-week recovery period.

There were no adverse effects on body weight, body weight gains, food consumption, or food efficiency, and there were no test substance-related or adverse clinical signs of toxicity in males or females exposed to any concentration over the course of this study.

No adverse effects on motor activity were observed in either males exposed to 1 or 10 ppm or female rats exposed to 1, 10, or 100 ppm. Male rats exposed to 100 ppm demonstrated decreased motor activity during the fourth week of exposure. The decreased motor activity in male rats exposed to 100 ppm had resolved following the 4-week recovery period. In the absence of other neurobehavioral changes in 100 ppm male rats or in female rats, the lower motor activity in 100 ppm males appears to be secondary to other systemic effects of the test substance, instead of a primary effect on the nervous system. There were no adverse effects observed in the ophthalmological evaluations at any exposure level in any of the animals in this study.

Following 4 weeks of exposure, concentration-related and adverse changes in clinical chemistry parameters were observed in male and female rats exposed to 100 ppm. In these animals, increased mean serum bilirubin levels were observed when compared to the control group and several females exposed to 100 ppm exhibited increased alanine aminotransferase (ALT). Following 4 weeks of recovery, group mean values for these clinical pathology parameters in both male and females were similar to the respective

control group and not statistically different. No effects on clinical chemistry parameters were observed in males or females exposed to 1 or 10 ppm.

There were no test substance-related adverse effects on gross pathological evaluation in male or females at any exposure level. Male and female rats exposed to 100 ppm demonstrated increased mean absolute liver weights as well as increased mean liver weights relative to body and brain weight one day following the last exposure. By the end of the recovery period, the liver weights were similar to control.

There were no test substance related adverse microscopic findings at any of the exposure concentrations tested. Test substance-related, but non-adverse, microscopic findings were present in the teeth (incisors) and sections of long bones (femur and tibia) at the termination of exposure and following the 1-month recovery period. These changes consisted of increased lamination of dentin of the incisor teeth and incomplete decalcification of enamel of the incisors and the bone trabeculae in the tibia and femur. These findings were consistent with exposure to a fluorine-containing test substance and were not associated with any histopathological changes suggestive of tissue injury or any adverse functional consequences in these tissues. Therefore, these microscopic findings were not considered to be adverse.

There were no portal of entry effects noted in this study; no adverse findings were observed in the respiratory tract tissues at any exposure level. Therefore, the adverse effects observed in this study are considered to be systemic effects and are consistent with those observed in a previous oral repeated dose study with the test substance.

Under the conditions of this study, the no-observed-adverse-effect concentration (NOAEC) for the test substance was 10 ppm for male and female rats based upon increased serum ALT and bilirubin levels (females) and increased liver weights in both male and females exposed to 100 ppm.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LOAEC
1 773 mg/m³
Study duration:
subchronic
Species:
rat

Additional information

A 28-day repeated dose oral toxicity study with the test substance is available. A 90-day repeated dose oral toxicity study and 28-day inhalation study with 6:2 FTOH were used as a read-across to fulfil the data gap for the test substance. The underlying hypothesis for the read-across between the test substance and 6:2 FTOH is that the test substance is transformed metabolically to 6:2 FTOH. The available in vitro data show that this transformation occurs extremely rapidly (half-life less than 3 minutes). While no actual in vivo rate data are available (aside from one study that demonstrates disappearance of the test substance), the transformation is still expected to occur rapidly. Additionally, based on the consistency in effects seen in the repeated dose studies coupled with the knowledge of the metabolism, it is reasonable to read-across the information from the 90-day oral and 28-day inhalation studies on 6:2 FTOH to address the data gap for the test substance.

Male and female rats were given 6:2 FTOH via gavage at doses of 0, 5, 25, 125, or 250 mg/kg/day for 90 days, with 1- and 3-month recovery periods. A NOEL of 5 mg/kg/day was established. At ≥25 mg/kg/day, changes were observed in the haematology and clinical chemistry parameters, urinalysis parameters (female rats only), urine fluoride and varied histopathological effects in the liver. Liver effects were initially (at the terminal sacrifice) varied but generally of minimal severity. They included single cell necrosis, vacuolation, oval/biliary hyperplasia, hepatocellular hypertrophy and periportal inflammation. Most liver findings disappeared by the 1 month recovery sacrifice and by 3 months only a few animals (125 and 250 mg/kg females) had biliary hyperplasia. Mortality was observed in one female rat at 125 mg/kg/day and six or more rats in each of the sexes at 250 mg/kg/day. Adverse clinical signs (primarily dental effects) were also increased in these groups, body weights and body weight changes were significantly reduced, plasma fluoride levels were increased and there was a test substance-related effect on the ameloblastic epithelium of the tooth. Feed consumption values were also significantly reduced in the female rats and increased apoptosis in the acinar cells at sacrifice in the male rats at 250 mg/kg/day. These alterations were generally reduced and/or not apparent at the 250 mg/kg/day dosage group after one month of recovery. Only increases in the liver and kidney organ weights observed in the female rats were apparent at ≥25 mg/kg/day at the 3-month recovery period, and a few female rats at 125 and 250 mg/kg/day were also observed with biliary hyperplasia in the histopathological evaluation. There were no biologically relevant effects on reproductive organs and tissues examined in male and female rats.

A 28-day repeated-dose oral toxicity study with a 14-day recovery period was performed with the test substance in male and female rats. Doses were 0, 1, 5, and 25 mg/kg/day, with recovery animals at 0 and 25 mg/kg/day to investigate the reversibility of effects. No test substance-related effects were observed on mortality, clinical signs, sensorimotor function, body weight, food intake, haematology, blood chemistry, or urinalysis. At the end of the dosing period, increased relative kidney weight in males, increased absolute and relative liver weights in females, and decreased iron pigment of the ameloblasts at maturation stage in the incisors were observed at 25 mg/kg. The changes in the incisor, kidneys, and liver were considered reversible, as after the recovery period, the liver and kidney weights were comparable to controls, and although mottled teeth were observed, no histopathological abnormalities were observed in the ameloblasts. The NOAEL for the study was set at 5 mg/kg/day, based on decreased iron pigment of the ameloblasts at maturation stage in the incisors in both sexes, increased relative kidney weight in males, and increased absolute and relative liver weights in females at 25 mg/kg/day. Although it was concluded that the NOAEL for the study was 5 mg/kg/day based on changes observed in rats in the 25/mg/kg/day group, these alterations may be considered test substance-related but not adverse. The increased relative kidney weight and absolute and relative liver weight in the 25 mg/kg/day group in males and females, respectively, were not associated with correlative histopathological changes or functional impairment of the organs as indicated by clinical pathology changes or systemic toxicity. Therefore, the kidney and liver weight changes in the 25 mg/kg/day group are considered minimal and adaptive. The histopathological changes observed in the incisors of some rats in the 25 mg/kg/day group, and the mottled appearance of the incisors in the 25 mg/kg/day group following the recovery period, are consistent with exposure to a fluorine-containing test substance and were not associated with any changes suggestive of tissue injury or any adverse functional consequence. Therefore, the microscopic findings in the incisors are not considered to be adverse. Although target organs were identified in the 28-day study, the duration and/or dosages were insufficient for the manifestation of adverse findings. Therefore, the 28-day study with the test substance is considered a supporting study.

6:2 FTOH was administered via gavage to Pgeneration male and female mice during premating, mating, gestation, and lactation, and to F1 generation male and female adults (post-weaning). P1 adult CD-1 mice (15/sex/group) were administered 6:2 FTOH daily in a vehicle of 0.1% Tween-80 in 0.5% aqueous methylcellulose at dosages of 0, 1, 5, 25, or 100 mg/kg body weight/day (mg/kg/day) for 10 weeks (males) and 2 weeks (females) during the premating period, and then up until the day before scheduled sacrifice.Adverse, test substance-related clinical signs and mortality were observed at 100 mg/kg/day in P1 males and females. In addition, test substance-related reductions in body weight, body weight gain, food consumption, and food efficiency were observed at this dose level in P1 males over the course of the study and in Pfemales during the lactation period. Adverse, test substance-related changes in haematology (red and white blood cell) and clinical chemistry (liver-related) parameters were present in P1 males and females administered 100 mg/kg/day. Changes in liver parameters were also present in one female mouse in the 25 mg/kg/day group. Red blood cell changes in the 100 mg/kg/day groups included minimal to mild decreases in red cell mass parameters (RBC, HGB, and HCT). These changes were slightly higher relative to controls in females compared to males, and in females, were associated with a regenerative response (increased ARET). White blood cell changes in the 100 mg/kg/day male and female groups included minimal to moderate increases in white blood cell parameters (WBC, ALYM, ANEU, and AMON). These changes were likely correlative to the liver effects observed at this dose level. Changes in liver-related clinical chemistry parameters in the 100 mg/kg/day male and female groups included elevations in AST, ALT, ALP, SDH, and TBA. These changes were greater in females compared to males, with elevations of greater than 5-fold for most parameters in females and less than or equal to 5-fold in males. Increases in these same liver parameters in one 25 mg/kg/day female mouse were associated with correlative test substance-related microscopic findings in the liver and were thus also considered test substance related and adverse. Other clinical chemistry changes observed in the 100 mg/kg/day male and female groups included decreases in BUN, CREA, and CHOL (males only). These changes were greater in males than in females and were considered to be secondary to the liver toxicity observed at this dose level. Adverse observations of anatomic pathology occurred in the liver and teeth of P1 males and females at 100 mg/kg/day and in the liver of females at 25 mg/kg/day. Liver changes were generally more severe in females and included hepatocellular hypertrophy, oval cell hyperplasia, single cell necrosis of hepatocytes, and cystic degeneration (females only). Increased liver weight was also observed at 100 mg/kg/day in males and females. In males and females at 100 mg/kg/day, microscopic changes in the incisor teeth consistent with fluoride exposure included degeneration and atrophy of ameloblastic epithelium, lamination of dentin, and incomplete decalcification of enamel and/or dentin. Changes considered test substance-related but non-adverse based on lack of association with organ injury or evidence of decreased function included increased kidney weights (100 mg/kg/day males), minimal hepatocellular hypertrophy (5 mg/kg/day males and females, 25 mg/kg/day males), and incomplete decalcification of enamel and dentin (25 mg/kg/day female group).The no-observed-adverse-effect level (NOAEL) for systemic toxicity was 25 mg/kg/day (males) and 5 mg/kg/day (females).

The key inhalation study is an OECD 412 compliant, 28-day inhalation study in rats with design concentrations of 0, 1, 10, and 100 ppm. Male and female rats exposed to 100 ppm of 6:2 FTOH demonstrated increased (~20-27%) mean absolute and relative (to body and brain) liver weights one day following the last exposure; by the end of the recovery period, the liver weights were similar to control. In females, 4 of 20 rats exposed to 100 ppm of 6:2 FTOH had ALT (a more specific and sensitive indicator of potential hepatic injury than AST and SDH) values more than double the historical and concurrent control values.  Bilirubin, another indicator of hepatic injury particularly in conjunction with increases in ALT, was minimally but statistically increased (~64% above controls) in 100 ppm females; absent other indicators, bilirubin values alone less than 2-fold above controls are considered to represent biological abnormalities rather than hepatic injury. However, only 1/20 females had an ALT and total bilirubin values more than two-fold above controls suggesting a very low incidence of hepatocellular injury at 100 ppm. In contrast, male rats had ALT values that were slightly increased but within the historical reference range. ALT and total bilirubin values were normal after a 4-week recovery. No histopathological changes were evident in the liver of males or females in any dose group. Nonetheless, the increased liver weights seen in both male and female rats at 100 ppm are considered adverse given the serum ALT levels observed. However, given that two or more enzymatic indicators of liver injury were increased by a factor or 2 or more compared to controls in only 1/20 females, 100 ppm can be considered to be a minimal effect level for hepatocellular injury. Since the clinical pathology changes at 100 ppm were of low incidence and absent histopathological correlates, the 100 ppm LOAEC should be used as the starting point rather than the 10 ppm NOAEC since the actual NOAEC is likely much closer to 100 ppm than 10 ppm.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
NOAEL of 5 mg/kg/day is based on read across to 1-generation reproduction study with 6:2 FTOH (Guideline, GLP study), which corrected for molecular weight differences between the test and read across substances, would yield an equivalent test substance NOAEL of 5.95 mg/kg/day.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
LOAEC of 1490 mg/m3 is based on read across to 4-week inhalation study with 6:2 FTOH (Guideline, GLP study), which corrected for molecular weight differences between the test and read across substances, would yield an equivalent test substance LOAEC of 1773 mg/m3.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; digestive: other

Justification for classification or non-classification

Based on the liver effects (weight, clinical pathology, and histopathological changes) observed in female mice at ≥25 mg/kg/day, as well as teeth effects (microscopic changes) observed in male and female mice at 100 mg/kg, after approximately 70 days of dosing in the 1-generation mouse gavage study with 6:2 FTOH, the test substance is classified as STOT Rep Cat 2 (H373: May cause damage to organs [liver; teeth] through prolonged or repeated oral exposure) according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.