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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
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).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
Remarks:
The study was conducted according to guideline in effect at time of study conduct.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 99.7%

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately 7 weeks.
- Weight at study initiation: 146 – 162 g (main and recovery males); 131 – 155 (main and recovery females); 154 – 182 g (satellite males); 143 – 161 g (satellite females)
- Housing: individually housed in stainless steel, wire-bottomed cages, except on the day before blood collection for the clinical pathology evaluation, at which time the rats will be placed in metabolism cages.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: approximately 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): targeted at 64°F to 79°F (18°C to 26°C)
- Humidity (%): targeted at 30% to 70%
- Air changes (per hr): Study rooms were maintained under conditions of positive airflow relative to a hallway and independently supplied with a minimum of ten changes per hour of 100% fresh air that had been passed through 99.97% HEPA filters.
- Photoperiod (hrs dark / hrs light): 12-hours dark: 12-hours light fluorescent light cycle.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% (w/v) aqueous methylcellulose prepared in reverse osmosis deionised water.
Details on oral exposure:
PREPARATION OF TEST SUBSTANCE FORMULATION:
Suspensions of the test substance were prepared at least once daily and the vehicle was prepared at least once weekly at the Testing Facility. Prepared formulations were stored at room temperature and used immediately. Prepared formulations were stirred continuously during dosage administration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of dose formulations were analysed for test substance by gas chromatography with flame ionisation detector (GC-FID). The method was validated for the analysis of dose formulations at concentrations ranging from 0.46 mg/mL to 50 mg/mL of test substance in 0.5% (w/v) aqueous methylcellulose.
The relative standard deviation of the mean of the average concentration values for the top, middle and bottom was calculated to assess homogeneity in the formulations prepared for use on the first day of dosage. Homogeneity was acceptable (≤ 5% relative standard deviation).
The average test substance concentrations in the samples of formulations prepared for use at the start, middle and end of the dosage period were within ±15% of the nominal concentrations with the following exceptions: the 1 mg/mL and 5 mg/mL (corresponding to the 5 and 25 mg/kg/day dosages, respectively) at the start of study were -42.4% and -19.7% of the nominal concentration, respectively; and the 1 mg/mL at the middle of the study was at -17.4% of the nominal concentration. Backup samples of the 1 mg/mL formulation from the first day of dosage as well as additional samples of all formulations prepared at the middle of the study were analysed at the sponsor's facility. These analyses confirmed that the 1 mg/mL formulation for the first day of study deviated substantially (measured concentration at this point was - 61.8% of the nominal value), but all concentrations were homogeneous. However, the average test substance concentrations in the samples of formulations prepared for use at the middle of the dosage period were within ±15% of the nominal concentrations.
Duration of treatment / exposure:
90 days (main study animals); 10 days (satellite animals)
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 5, 25, 125, and 250 mg/kg/day (males and females)
Basis:
other: actual ingested (main study animals)
Remarks:
Doses / Concentrations:
0, 5, 25, 125, and 250 mg/kg/day (males and females)
Basis:
other: actual ingested (satellite animals)
No. of animals per sex per dose:
Ninety-five male and ninety-five female rats were randomly assigned to five dosage groups (Groups I through V), 10 rats per sex per group, using a computer generated (weight-ordered) randomisation procedure. An additional ten rats per sex in Groups I and V were assigned to the 1-month recovery phase of the study and an additional five rats per sex in Groups I through V were assigned to the 3-month recovery phase of the study. Rats assigned to the 10-day satellite portion of the study were assigned to five dosage groups (Groups I through V), five rats per sex per group, using a computer-generated (weight-ordered) randomisation procedure; the satellite rats were designated for future biochemical analyses.
See Table 1 and Table 2
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels used on this study were 0 (Vehicle), 5, 25, 125 and 250 mg/kg/day. These doses were selected based on the results from previously conducted studies with chemically similar test substances in which similar dose levels were tested; the doses tested ranged from 1 to 250 mg/kg/day. Test substance-related effects varied between the three previous studies in specific terms; however, in general terms, the lowest effect levels ranged from 25 to 125 mg/kg/day. Effects observed at these doses included reductions in body weight and food consumption parameters and clinical and necropsy observations, including effects on teeth and effects on the liver and kidneys. The no-effect levels in the previous studies ranged from 5 to 25 mg/kg/day. Previous relevant work with the current test material has indicated that the oral LD50 in rats is 1750 mg/kg/day. In the current study, the high dose level of 250 mg/kg/day was expected to produce evidence of toxicity; whereas, the high-intermediate dose level of 125 mg/kg/day was expected to be slightly less toxic. Based on these expectations, toxicity was not expected at 25 mg/kg/day or at the low dose of 5 mg/kg/day.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: For viability at least twice each day. For clinical observations of effects of the test substance before and one to two hours after dosage administration (for each animal), and once daily during the postdosage period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At the end of the first week of dosage administration and once weekly thereafter. Observations such as localised alopecia, sparse hair coat and increased motor activity were recorded. Signs noted included, but were not limited to sparse hair coat, localised alopecia, soft or liquid faeces, rales, and misaligned, broken or missing incisors.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during the dosage period and on the day before sacrifice. Body weights were also recorded weekly during the postdosage period (recovery rats only) and on the day of sacrifice (terminal weight).

FOOD CONSUMPTION; Yes
- Time schedule: Weekly during the dosage period and the postdosage period (recovery rats only) and on the day before sacrifice (feed left value).

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations and dose groups examined: Prior to dosage on all rats (except those designated for the 10-day hepatic biochemical evaluation). Any rats with preexisting ophthalmological abnormalities were eliminated from consideration for use on the study. Surviving rats in the main study portion (10 rats/sex/dosage group if possible) were examined on study days 82 to 85.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during week 7 and 14 (prior to necropsy). At necropsy following the completion of the 3-month recovery period
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: Yes
- How many animals: all rats (main study and recovery animals)
- Parameters checked in Table 3 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during week 7 and 14 (prior to necropsy) and at necropsy following the completion of the 3-month recovery period. An additional 2.0 mL sample of whole blood was taken from main and recovery rats at week 14 of study and after 1 and 3 months of recovery for analyses of fluoride concentration.
- Animals fasted: Yes
- How many animals: all rats (main study and recovery animals)
- Parameters checked in Table 4 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: during weeks 7, 9 and 14. An additional urine sample was taken from each rat assigned to the main and recovery portions of the study during week 9 of the study for analyses of fluoride concentration.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in Table 5 were examined.
Sacrifice and pathology:
GROSS PATHOLOGY/ HISTOPATHOLOGY: Yes

Satellite animals:
Rats were fasted after 3 PM (at least 15 hours) on the afternoon before their scheduled sacrifice. Rats were sacrificed by carbon dioxide asphyxiation and exsanguination on the day following the last administration of the test substance and/or vehicle. A gross necropsy was performed. At the time of sacrifice, liver samples were collected for possible future evaluation. Rats were discarded without further evaluation.

Main study and 1-month and 3-month recovery animals:
All surviving male and female rats were sacrificed by carbon dioxide asphyxiation and examined for gross lesions on the day following the last administration of the test substance and/or vehicle or at the end of the 1-month and 3-month recovery periods. Gross necropsy included an initial physical examination of external surfaces and all orifices, as well as an internal examination of tissues and organs in situ. In addition, the cranial, thoracic and abdominal cavities were examined. The lungs were perfused with neutral buffered 10% formalin. Gross lesions were retained in neutral buffered 10% formalin and examined histologically. Bone marrow was collected from the sternum and duplicate bone marrow smears were prepared at the time of sacrifice from all surviving rats and were evaluated for quality by visual examination. At the time of sacrifice, blood (then processed to plasma), liver and fat samples were collected from all rats assigned to the main study and 1 and 3 month recovery periods for analyses of test substance and/or metabolite. The test substance, and proposed metabolites, PFBuA (F(CF2)3COOH), PFPeA (F(CF2)4COOH), PFHxA (F(CF2)5COOH), PFHpA (F(CF2)6COOH), 5-3A (F(CF2)5CH2CH2COOH), 6-2A (F(CF2)6CH2COOH), 6-2UA (F(CF2)5CF=CHCOOH), and 4-3A (F(CF2)4CH2CH2COOH) were quantified by LC/MS/MS analysis.

Organs collected and weighed: see Table 6

Tissues collected: see Table 7
Histological examination was performed on all control and high test substance concentration group rats, and all target tissues from all rats. All tissues with lesions considered test substance-related in the high test substance concentration were examined histologically in the rats exposed to the lower test substance concentrations. All gross lesions were examined histologically.

Rats that died or were sacrificed before scheduled termination were examined for the cause of death or condition on the day the observation was made. The rats were necropsied and examined to the extent possible. Tissues (see Tables 6 and 7) were weighed and retained for histological evaluations.

Statistics:
All data were tabulated, summarised and/or statistically analysed using the Argus Automated Data Collection and Management System, the
Vivarium Temperature and Relative Humidity Monitoring System, Microsoft Excel (part of Microsoft® Office 97/2000/2003/XP), Quattro Pro 8 and
The SAS System (version 6.12). Statistical evaluations of data were done using parametric methods for normally distributed data and nonparametric methods for count data.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
males only
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
females only
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
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:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY
Males:
At 250 mg/kg/day, there was a significant increase in the number of male rats that were either found dead or euthanised because of adverse clinical signs, generally after 56 to 81 dosages. Most of these deaths were attributed to degeneration and necrosis in the kidneys. Three male rats were also observed with moderate to marked haemorrhage and acute inflammation in the nose.
Dental effects were observed at 250 mg/kg/day during the dosage period. Relative to the vehicle control group values, significantly more rats in the 250 mg/kg/day dosage group had excess salivation, urine-stained abdominal fur, dehydration (based on skin turgor) and perioral substance. The dental effects noted during the dosage period persisted into the recovery period, and were noted in a significantly increased number of male rats at ≥ 125 mg/kg/day.
The adverse clinical signs observed during the detailed clinical observations were consistent with those observed during the standard clinical observations.
Females:
Mortality occurred at 125 and 250 mg/kg/day. At 125 mg/kg/day, one female rat was sacrificed due to adverse clinical signs. There was also a significant increase in the total number of female rats at 250 mg/kg/day that were either found dead or sacrificed due to adverse clinical signs, generally after 19 to 73 dosages. Most of these deaths were attributed to degeneration and necrosis in the kidneys.
Dental problems were observed at 125 and 250 mg/kg/day during the dosage period. Significantly more female rats at 125 and 250 mg/kg/day had urine-stained abdominal fur, piloerection and scant faeces, while excess salivation, dehydration (based on skin turgor), hunched posture, coldness to the touch, ungroomed coat, decreased motor activity and pale extremities were noted in significantly more female rats at 250 mg/kg/day. The dental effects noted during the dosage period persisted into the recovery period, and were noted in a significantly increased number of male rats at 125 and 250 mg/kg/day.
The adverse clinical signs observed during the detailed clinical observations were consistent with those observed during the standard clinical observations.

BODY WEIGHT AND WEIGHT GAIN
Males:
At 125 and 250 mg/kg/day average body weight gain was 14% and 11% lower, respectively, than the vehicle control group value. Interrelated with the reductions in body weight gains, mean body weights at the end of the dosage period were significantly reduced at ≥ 125 mg/kg/day, and remained reduced or significantly reduced during the recovery period.

FOOD CONSUMPTION AND COMPOUND INTAKE
Females:
At 250 mg/kg/day, absolute and relative feed consumption values were lower or significantly lower during the entire dosage period.

HAEMATOLOGY
Males:
At 250 mg/kg/day significant reductions in haemoglobin and haematocrit levels occurred on day 43 of study. At the end of the dosage period, red blood cell counts, haemoglobin and haematocrit levels were lower or significantly lower at ≥ 25 mg/kg/day, and mild, but statistically significant reductions in the activated partial thromboplastin time occurred in these same dosage groups. These haematological changes did not persist during the recovery period.
Females:
At ≥ 125 mg/kg/day red blood cell counts, haemoglobin levels and haematocrit levels were significantly lower relative to vehicle control group values on day 43 of study. In addition, the percent red cell distribution width, percent reticulocytes and absolute reticulocytes were increased in these same dosage groups. These findings, with the exception of the changes in reticulocytes, were consistent with observations noted at the end of the dosage period. At dosages of 125 or 250 mg/kg/day there were mild, but statistically significant reductions in the activated partial thromboplastin time at the end of the dosage period.

CLINICAL CHEMISTRY
Males:
At the end of the dosage period, changes in serum chemistry included an increase or significant increase in the albumin/globulin ratio at ≥ 25 mg/kg/day; and the inorganic phosphorus, protein, albumin, total bilirubin and potassium at ≥ 125 mg/kg/day. There was also a reduction or significant reduction in blood urea nitrogen levels at ≥ 125 mg/kg/day. By the end of the recovery period, these changes in serum chemistry had resolved.
Females:
Changes in serum chemistry included significantly increased cholesterol at ≥ 25 mg/kg/day; and albumin, total bilirubin and triglyceride levels at ≥ 125 mg/kg/day; and creatinine levels, blood urea nitrogen levels, calcium levels and inorganic phosphorus levels at 250 mg/kg/day. By the end of the dosage period, most of the changes in clinical chemistry had resolved with the exception of the changes in total bilirubin levels. Additional findings that occurred at the end of the dosage period included an increase or significant increase in cholesterol levels at ≥ 125 mg/kg/day, and elevations in aspartate aminotransferase and alkaline phosphatase at ≥ 25 mg/kg/day. The elevations in aspartate aminotransferase and alkaline phosphatase were consistent with changes in liver weights that occurred in these same dosage groups.
Males and Females:
Plasma fluoride levels were increased and statistically significant at 25 mg/kg/day (males) and ≥ 125 mg/kg/day (females). Following approximately one month of recovery, plasma fluoride at 250 mg/kg/day was still increased but had partial recovery. Plasma fluoride levels were similar to controls following approximately 3 months of recovery.

URINALYSIS
Females:
Significantly more female rats at ≥ 125 mg/kg/day had trace amounts of blood present in the urine at day 63 of study. At ≥ 25 mg/kg/day there was a statistically significant reduction in the number with blood absent in the urine. At the end of the dosage period, the specific gravity of the urine at 25 and 125 mg/kg/day were significantly lower, and there were trace amounts of protein present in the urine at ≥ 125 mg/kg/day.
Males and Females:
Urine fluoride was increased in a dose-related manner in all treated dosage groups (statistically significant in the ≥ 25 mg/kg/day dosage groups) during the dosing period at both the week 9 and day of study 91 time points.

ORGAN WEIGHTS
Males:
The absolute and relative (% brain weight) weight of the liver and the paired kidneys was increased or significantly increased at ≥ 125 mg/kg/day at the end of the dosage period. Relative to body weight, these organs were significantly increased at ≥ 25 mg/kg/day at the end of the dosage period, and residual effects were noted at 250 mg/kg/day one month after the completion of the dosage period. These observations were not present three months after the completion of the dosage period. In addition, the relative weights of the paired testes and paired epidydimides was significantly increased at ≥ 25 mg/kg/day.
Females:
The absolute and relative (% body weight and % brain weight) weight of the liver and the paired kidneys was increased or significantly increased at ≥ 25 mg/kg/day at the end of the dosage period. One month after the completion of dosage administration, the weights of the liver and paired kidneys remained significantly increased at 250 mg/kg/day. In general, residual effects were noted in these organs three months after the completion of the dosage period at ≥ 25 mg/kg/day; however, these increases did not reach statistical significance.

GROSS PATHOLOGY
Males and Females:
At ≥ 125 mg/kg/day white discoloration of the teeth was observed in terminal sacrifice rats. This observation persisted in the 1-month 250 mg/kg/day recovery rats but had nearly disappeared (or was at low incidence in the limited number of rats sacrificed) at the 3-month time point.

HISTOPATHOLOGY: NON-NEOPLASTIC
Males:
Histopathological effects in the liver were observed at terminal sacrifice at 125 (single cell hepatocellular vacuolation) and 250 mg/kg/day (hepatocellular hypertrophy, single cell necrosis, biliary hyperplasia, periportal inflammation and hepatocellular vacuolation). At 250 mg/kg/day, effects on the teeth (ameloblastic dysplasia and degeneration or attenuation of the ameloblastic epithelium) were also observed at terminal sacrifice. Acinar cell apoptosis was also observed at 250 mg/kg/day. The liver findings were no longer apparent by the 1-month recovery sacrifice. Although decreased, effects on the teeth (ameloblastic dysplasia and enamel dysplasia) and acinar cell apoptosis were apparent at the 1-month recovery sacrifice.
Females: Histopathological effects in the liver were observed at terminal sacrifice at 25 (oval cell hyperplasia) and 125 mg/kg/day (single cell necrosis and oval cell hyperplasia). There was only one female rat remaining at terminal sacrifice in the 250 mg/kg/day dosage group and there were no histopathological effects observed in this female. At 250 mg/kg/day, the liver findings (oval cell hyperplasia and periportal pigmentation), effects on the teeth (loss of ameloblastic epithelium and enamel dysplasia) and acinar cell apoptosis were all apparent at the 1-month recovery sacrifice. The only finding present at the 3-month recovery sacrifice was a few female rats in the 125 and 250 mg/kg/day dosage groups with residual biliary hyperplasia.

Effect levels

Dose descriptor:
NOEL
Effect level:
5 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Changes in haematology and clinical chemistry parameters, urinalysis parameters (female rats only), urine fluoride and varied histopathological effects in the liver at 25 mg/kg/day and higher.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Based on the toxicokinetic evaluation, the plasma, liver and fat samples displayed that 5-3A (F(CF2)5CH2CH2COOH) was the major metabolite observed at all dose levels and in both sexes. The 4-3A (F(CF2)4CH2CH2COOH) and PFPeA (F(CF2)4COOH) metabolites were also observed but in much lower concentrations than 5-3A. Liver- or fat-to-plasma ratios did not indicate preferential retention in these organs. Using the plasma concentrations of 5-3A as a marker for internal exposure shows that female rats have a higher internal dose than the male rats. Male rats reached saturation at the 125 mg/kg/day dose level. It is likely that the presence of 5-3A, 4-3A and PFPeA in the 1- and 3-month recovery samples reflects the biphasic elimination common to this class of test materials. This biphasic elimination is characterized by an initially rapid elimination of the majority of the test material followed by a slower elimination of a small fraction.

Applicant's summary and conclusion

Conclusions:
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).

NOEL = 5 mg/kg/day
Executive summary:

The purpose of this study was to provide information on possible adverse effects on rats resulting from repeated exposure to the test substance over an extended period of time (90 days) covering postweaning maturation and growth well into adulthood. Ten rats per sex per group were dosed at 0, 5, 25, 125, and 250 mg/kg/day. Additional satellite groups included 1-month, 3-month, and 10-day groups. Based on the results of this study, the no-observable-effect-level (NOEL) for toxicity of the test substance is 5 mg/kg/day. At 25 mg/kg/day and higher dosages, changes were observed in the haematology and clinical chemistry parameters, urinalysis parameters (female rats only), urine fluoride and varied histopathological effects in the liver. At 125 and 250 mg/kg/day, 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 effects 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 in the ten rats per sex in the 250 mg/kg/day dosage group available for examination after one month of recovery. Among the five rats/sex/dosage group examined after the 3 -month recovery period, only increases in the liver and kidney organ weights observed in the female rats were apparent at the 25 mg/kg/day and higher dosages, and a few female rats at 125 and 250 mg/kg/day were also observed with biliary hyperplasia in the histopathological evaluation. Based on the toxicokinetic evaluation, the plasma, liver and fat samples displayed that 5-3A (F(CF2)5CH2CH2COOH) was the major metabolite observed at all dose levels and in both sexes. The 4-3A (F(CF2)4CH2CH2COOH) and PFPeA (F(CF2)4COOH) metabolites were also observed but in much lower concentrations than 5-3A. Liver- or fat-to-plasma ratios did not indicate preferential retention in these organs. Using the plasma concentrations of 5-3A as a marker for internal exposure shows that female rats have a higher internal dose than the male rats. Male rats reached saturation at the 125 mg/kg/day dose level. It is likely that the presence of 5-3A, 4-3A and PFPeA in the 1- and 3-month recovery samples reflects the biphasic elimination common to this class of test materials. This biphasic elimination is characterized by an initially rapid elimination of the majority of the test material followed by a slower elimination of a small fraction.