2-Propenoic acid, γ-ω-perfluoro-C8-14-alkyl esters

Repeated dose toxicity: oral

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

Study period:

not mentioned in publication

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study is a scientific publication and well documented. The read across hypothesis is based on the assumption that due to its characteristic as ester, after ingestion in mammalians the ester bound of the target chemical will be subject to metabolic cleavage into its corresponding alcohols and the acrylic acid. Thus Fluowet AC 812 (target) and its associated alcohols (perfluoro-alkylethanol) n:2 FTOH mixture are considered to form not only a group of structurally related analogues, but are also closely related based on metabolic pathway considerations. For details see section 13 of this IUCLID document

Data source

Reference

Reference Type:

publication

Title:

Subchronic Toxicity of a Fluoroalkylethanol Mixture in Rats

Author:

Ladics, Gregory S.

Year:

2005

Bibliographic source:

Drug and Chemical Toxicology, 28:135-158

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Fluoroalkylethanol mixture

IUPAC Name:

Fluoroalkylethanol mixture

Constituent 2

Reference substance name:

65530-60-1

EC Number:

613-814-1

Cas Number:

65530-60-1

IUPAC Name:

65530-60-1

Test material form:

other: pale to medium tan waxy solid

Details on test material:

Analytical purity: 95%
- Impurities (identity and concentrations): about 2% residual fluoroalkylethyl jodide (i.e. chemical precursor to the fluorotelomer alcohol)
Water and other unidentified impurities composed the remainder.
- Composition of test material, percentage of components: FTOH mixture F(CF2CF2)nCH2CH2OH, n= 3 to 6
- Purity test date: n.a.
- Lot/batch No.: n.a.

Test animals

Species:

rat

Strain:

other: CrJ:CD(SD)IGS BR rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (Raleigh, NC,USA)
- Age at study initiation: approx. 7 weeks
- Weight at study initiation: males: about 250 g (only presented as first point of a graph, demonstrating the body weight gain over time covering the complete study period)
- Housing: All rats were housed one per cage, sexes separate, in stainless steel, wire-mesch cages
- Diet: ad libitum. PMI Nutrition International, Inc., Certified Rodent LabDiet 5002. Initially all rats were fed pelleted chow; however, beginning on test day 57, animals exhibiting effects to the teeth were placed on ground chow. On test day 77, all other rats in the 250mg/kg/day dose group were placed on ground chow.
- Water: tap water ad libitum
- Acclimation period: about 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): n.a.
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark cycle

IN-LIFE DATES: not applicable

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous methylcellulose

Details on oral exposure:

Dosing suspensions were prepared daily prior to dosing with 0.5% aqueous methylcellulose from test day 0 to day 37 (with a dose volume of 10 mL/kg). On test day 38 the dosing volume for male and female rats was decreased from 10 mL/kg to 7.5 mL/kg. As a result of stability testing that indicated refrigerated dosing suspensions were stable for 4 days, dose suspensions were prepared twice each week starting on test day 42 until the end of the dosing period.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily by gavage

Doses / concentrations

Remarks:

Doses / Concentrations:
The dosages were 0, 25, 100, or 250 mg/kg/day
Basis:
other: Test item was prepared to achieve 0, 25, 100, or 250 mg active ingredient per kg body weight per day, based on the most recently recorded body weight.

No. of animals per sex per dose:

High-dose and control-dose group: 30/rats sex
Intermediate and low dose group: 15 rats/sex.

Control animals:

yes, concurrent vehicle

Details on study design:

High-dose and control-dose group:
There was a total of 25 rats/sex assigned to the high-dose and control groups and 15 rats/sex to the intermediate and low dose groups.
Ten male and 10 female rats in each group were dosed up to 90 days and necropsied.
Ten male and 10 female rats of the high- and control-dose groups were designated for 1 month recovery evaluation.
5 rats /sex in each dose group were designated as a 3-month recovery subset.
Additional 5 rats/sex per dose were received separately and designated for biochemical evaluation (i.e. hepatic beta-oxidation) after a 10 day exposure.

Neurobehavioral evaluations were conducted on male and female rats designated for the 90 day exposure and on control and high-dose animals designated for the 1-month recovery.
Biochemical evaluations (i.e. hepatic beta-oxidation) were performed on animals after 10 and approx. 90 days of dosing and after 1- and 3-month recovery periods.
Clinical pathology evaluations were conducted on animals designated for the 90 day exposure evaluations on weeks 7 and 13 and on animals designated for the 1-month recovery evaluation, immediately prior to necropsy.

Examinations

Observations and examinations performed and frequency:

BBODYWEIGHT AND CLINICAL OBSERVATION.
Rats were weight once per week during the exposure phase. In addition, the rats designated for neurobehavioral evaluations were weighed on the days of these observations. During the test period, cage-site examinations were conducted at least twice daily. Detailed clinical observation in a standardized arena were also evaluated on rats designated for the 90-day exposure and 1-month recovery periods.

FOOD CONSUMPTION:
The amount of food consumption was determined weekly. From these measuremnets, mean daily food consumption over the interval was determined. From the food consumption and bodyweight data the mean daily food efficiency was calculated. No detailed data presented but effects on food consumption are discussed in the result part.

FOOD EFFICIENCY:
No data presented

WATER CONSUMPTION:
No data presented

OPHTHALMOSCOPIC EXAMINATION: No data presented

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples from the orbital sinus were collected during week 7 and 13 and at 1 month of recovery
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 10 animals per sex and per group
- Parameters checked: Only red blood cell mass parameters, i.e. red blood cells, hemoglobin and hematocrit are mentioned in the publication. No other hematology parameter are presented in the paper.

BLOOD AND URINE FLUORINE Assays
Total Fluorine in Whole blood
In peliminary studies whole blood samples were collected into EDTA tubes approximately once each week from 5 rats group/sex dosed daily with either 5 or 50 mg/kg/day of test substance (data of a 45-day treatment are presented as figure in the paper). Blood was stored and refrigerated until analysed. During the main study plasma was evaluated for presence of fluoride in its ionic form only [F-]. The total fluorine content of the blood samples was de termined by usinf a Wickbold torch combustion method (Capka et al. 2004). Limit of detection (LOD) for this method is 0.5 ppm (500ppb)
Plasma Fluoride:
Blood for plasma anylysis was collected from the vena cava at sacrifice. Plasma fluoride concentration was determined on EDTA Plasma at the end of treatment and the 1-month recovery period using phi/12 pH meter with fluoride-selective electrode.
Urine Fluoride:
Overnight urine samples were used for the determination of urine fluoride. Urine fluoride was determined by multiplication of measured urine volume by urine fluoride concentration (measured by using phi/12 pH meter with fluoride-selective electrode). Urine fluorides were determined at the end of exposure period and at the end of the 1-month and 3-month periods.

CLINICAL CHEMISTRY: Yes, no data presented within the paper

BIOCHEMICAL MEASUREMENT:
- Time schedule for collection of blood: Blood samples from the orbital sinus were collected during week 7 and 13 and at 1 month of recovery
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 10 animals per sex and per group
- Parameters checked: hepatic beta-oxidation was examined [table 2].

URINALYSIS: Yes
- Time schedule for collection of urine: End of treatment (overnight urine samples).
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked: Fluoride. At the end of exposure period urine fluoride was determined by total fluoride excreted over a 24-h period. No other urine data presented.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to study start, during week 13 and after the one month recovery period.
- Dose groups that were examined: All groups at study start and during week 13. Control and high dose group after one month recovery.
- Battery of functions tested: sensory activity / grip strength / motor activity .

Sacrifice and pathology:

At the end of the exposure period, rats were randomly sacrificed by carbon dioxide anesthesia and exsanguination and necropsied.
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Representative samples of the following tissues were saved from the 90-day exposure and 1-month recovery groups: liver, kidneys, lungs, duodenum, heart, aorta, spleen, brain (cerebrum, midbrain, cerebellum, medulla/pons), spinal cord (three levels: cervical, mid-thoracic, lumbar), stomach, jejunum, ileum, pancreas, cecum, colon, rectum, mesenteric and mandibular lymph nodes, thymus, adrenal glands, sciatic nerve, thyroid gland, parathyroid glands, trachea, esophagus, pharynx/larynx, eyes (including optic nerve); prostate, seminal vesicles, urinary bladder, testes, epididymides, mammary glandy, ovaries, uterus, mandible, sternum (with bone marrow), femur/knee joint (with bone marrow), nose, salivary glands, pituitary gland, skeletal muscle and skin.
The following tissues were collected and weighed (except nose) from 3-month recovery group rats: Liver, kidneys, thyroid gland (after fixation), testes and nose.
All tissues collected from the control and 250 mg/kg/d rats that were sacrificed at the end of exposure period were examined microscopically. Liver, kidneys, thyroid gland, nose and mandible were identified as possible target organs and were processed to slides and examined microscopically in the 25 and 10 mg/kg/d 90-day exposure and 1- month recocvery groups.
Liver, kidneys, thyroid gland, and nose from males and thyroid gland and nose from females in the 3-month recovery group rats were also processed to slides and examined microscopically.

Other examinations:

Observed abnormalities in teeth were specifically examined by histopathological screeening of the enamel organ ameloblast cells.

Statistics:

Yes.
Dunn's test; Dunnett/Tamhane-Dunett test; Jonckheere-Terpstra trend test;

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

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

effects observed, treatment-related

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not specified

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

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No test substance related mortality occurred in the study.
A test substance related increase in the incidence of broken (males) and absent (males and females) teeth was observed in the 250 mg/kg/day dose group. The abnormalities in teeth were first observed on study day 58 in the 250 mg/kg/day dose male group and subsequently began to increase in incidence. By the end of the respective dosing phases, 10/25 males and 1/25 females in the 250mg/kg/d dose groups had broken teeth and 2/25 males and 1/25 females had teeth that were missing. Observations regarding teeth in the 25 and 100mg/kg/d dose groups were unremarkable (no broken teeth, one male in the 100mg/kg/d dose group with missing tooth).

BODY WEIGHT AND WEIGHT GAIN
The abnormalities in teeth were first observed on study day 58 in the high dose male group and subsequently began to increase in incidence. These observation were coupled with statistically significant decrements in body weight gain by day 56 (6-14% and 6-12 % in males and females, respectively). By day 77, males and females in the 100mg/kg/d dose group also had lower body weight gain (7-8% and 6-7%, respectively). Observations regarding teeth in the 25 and 100mg/kg/d dose groups were unremarkable (no broken teeth, one male in the 100mg/kg/d dose group with missing tooth). No effects on body weight were seen in male or female rats dosed with 25mg/kg/d. After day 77, rats in the 250mg/kg/d male and female groups were given ground chow rather than the pelleted type offered initially. A significant increase in body weight gain followed for these groups. However, the magnitude of increase in body weight gain for the 250mg/kg/d group is uncertain, as animals in the other groups may have experienced similar increases in bodyweight gain had thay placed on ground chow. Male and female weight gain responses very similar. Overall, for the subchronic study significantly lower body weight gains of 13% and 16% occurred in the 100 and 250mg/kg/d male groups, respectively, and 12% in both the 100 and 250mg/kg/d female groups.
After 1-month recovery period, the mean body weight and body weight gain of both male and female rats received 250mg/kg/d approached control values but were still significantly less. After 3-month recovery period, the mean body weight (female only) and body weight gain of both males and females exceeded control levels (data not shown in the paper).

FOOD EFFICIENCY
Effects on food consumption were noted in the 100 and 250mg/kg/d dose groups and were closely correlated to body weight gains (data not shown in the paper). After a 1-month recovery period, mean food consumption in the 250mg/kg/d male group was still lower, whereas for females, food consumption was similar to control. After 1 month of recovery, mean food consumption for males and females was similar to controls.

OPHTHALMOSCOPIC EXAMINATION
No data

HAEMATOLOGY
Male and female rats dosed with 250mg/kg/d had minimally decreased red cell mass parameters (red blood cells (RBC), hemoglobin, hematocrit); variable statistical significance after 45 and 90 days. These decrements were associated with minimally increased red cell distribution width (RDW) in both sexes; anisocytosis, microcytes, and increased reticulocytes in males; and poikilocytosis in females. After the 1-month recovery period, male rats previosly dosed with 250mg/kg/d still had minimally to mildly decreased red cell mass parameters and decreased reticulocytes compared to control group rats. Female rats previously dosed with 250mg/kg/d had red cell mass parameters similar to controls at the end of the 1-month recovery period, although mean corpuscular volume, mean corpuscular hemoglbin RDWW, and reticulocytes were decreased.

CLINICAL CHEMISTRY
There were no toxicologcally significant changes in clinical chemistry parameters in rats dosed at any dose level. Biochemical evaluation of hepatic beta-oxidation was performed in liver peroxisomes. Dose-dependant increases in the rate of hepatic beta-oxidation were observed at the 10-day time point with statistical significance occurring for rats administered 100 and 250 mg/kg/d (223% and 349% of control in the males and 170% and 260% of control in the females, respectively).
Dose-dependant increases in the rate of hepatic beta-oxidation were observed at the 90-day time point with statistical significance occurring for rats administered 100 and 250 mg/kg/d (405% and 872% of control in the males and 345% and 381% of control in the females, respectively). At both time points the increases in hepatic beta-oxidation activity were accompanied by increases in liver weights. No effect on biochemical end points was observed for rats administered 25mg/kg/d. Following 1 month recovery period, statistically significant increases in the rate of hepatic beta-oxidation persisted in rats administered 250mg/kg/d (353% and 207% of control, respectively, in males and females). Statistically significant increases in the rate of hepatic beta-oxidation also persisted after 3-month recovery period for the 250mg/kg/d dose group (302% and 172% of control, respectively, in males and females). At the 1-month recovery time point, the increase in hepatic beta-oxidation activity was accompanied by increased liver weights in the males not the females.

BLOOD FLUORINE Assays
Whole blood:
During peliminary range finding studies whole blood samples were collected approximately once each week from 5 rats group/sex dosed daily with either 5 or 50mg/kg/day of test substance and were analysed for total fluorine (data of a 45-day treatment are presented as figure in the paper). These measurements represented both organic fluorine and fluoride ion present in circulating blood (plasma and cellular). Measurements from blood samples taken approximately once per week indicated that the concentration of fluorine increased with duration of dosing. An apparent steady state was achieved after approximately 40-45 days, and levels of bloodfluorine were generelly higher for males than for females.
Plasma:
During the present study plasma was evaluated for presence of fluoride in its ionic form only [F-]. This plasma fluoride was mildly increased in males and females dosed with 100 or 250mg/kg/d at the end of the 90-day exposure period. After a 1-month recovery, plasma fluoride in 250mg/kg/d rats was similar to control group values. Plasma levels were not evaluated thereafter.

URINALYSIS
At the end of the exposure period, urine fluoride (as determined by total fluoride excreted over a 24-h period) was increased in a dose dependent manner in males and females administered 25, 100, or 250mg/kg/d. Following a 1-month recovery, daily urine fluoride excretion was increased in mals and females previously treated with 250mg/kg/d, although levels were markedly reduced relative to those present at the end of exposure. After a 3-month recovery period, urine samples from rats previosly dosed with 250mg/kg/d still contained significant levels of fluoride, although levels were reduced relative to those at the end of the 1-month recovery period. In addtion, urine fluoride was still increased over control group values for males and females previously dosed wi th 100mg/kg/d (males statistically significant).

NEUROBEHAVIOUR
No adverse changes in neurobehavioral parameters were observed in males and females in any dose group.

ORGAN WEIGHTS
Increase in liver and kidney weight parameters in rats dosed with 25 (liver: males only), 100 and 250 mg/kg/d for 90 days. Increase correlated with microscopic changes in liver and kidneys (m) and persisted in the high dose 1-month recovery group.

HISTOPATHOLOGY: NON-NEOPLASTIC
Degeneration /disorganization of enamel organ ameloblast cells occurred in both male and female rats administered 100 or 250mg/kg d
Liver:
Statistically significant increases in liver weight parameters were present in males and females administered 25 (males only) 100 , or 250mg/kg/d for 90 days. The increased liver weight correlated with microscopic centrilobular hepatocellular hypertrophy in the 100 and 250mg/kg/d male and female groups. Increased liver weights persisted in the 250mg/kg/d male and female groups through 1 month of recovery, although the magnitude was decreased compared to the end of the exposure period. Hepatocellular hypertrophy persisted with decreased severity in male rats. After 3 months, liver weights remained increased in both the 250mg/kg/d male and female groups, although the magnitude was less than that observed after end of the dosing period. Hepatocellular hypertrophy was observed only in the 250mg/kg/d male group (1 of 5 rats).
Kidney:
Statistically signifcant increases in kidney weights also occurred in males and females administered 25mg/kg/d or more. The increase kidney weights correlated with microscopic renal tubular hypertrophy in the 100 (1 of 10 rats) and 250mg/kg/d male groups only. There were no microscopic correlates to kidney changes in females. One month postdosing, the renal tubular hypertrophy persisted with decreased incidence and severity in males, while some kidney weight parameters remained increased in both male and female rats. No alterations in kidney weight parameters or renal tubular hypertrophy were observed at any dose level after 3 month of recovery.
Thyroid:
Increases in thyroid hypertrophy were observed in the 100 and 250mg/kg/d male and 250mg/kg/d female dose groups. Thyroid hypertropy was minimal and unassociated with proliferative thyroid lesions. In addition, alterations in thyroid colloid were observed in male and female rats at all dose levels. After 1 month recovery, the thyroid hypertrophy was no longer observed, although alterations in thyroid colloid persisted in both male and female rats. Alterations in thyroid colloid were still present in both male and females after 3 month of recovery.

OTHER FINDINGS

Effect levels

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The selected NOAEL of 25mg/kg/d is based on increased hepatic peroxisomal beta-oxidation, and the degeneration and/or disorganization of enamel organ ameloblast cells in males and females administered 100 mg/kg/d. In males additionally body weight decrements were observed which could be attributed to alterations to the teeth caused by fluorosis and therefore, decreased ability of animals to eat pelleted chow. The NOAEL of 25 mg/kg/d is also predicted for the substance registered.

Executive summary:

In the subchronic toxicity study rats were administered an FTOH mixture [F(CF₂)_xC₂H₄OH, x = 3 to 6] by oral gavage at 0, 25, 100 or 250 mg/kg/d. One- and three-month recovery groups were also included to assess the reversibility of any toxic effects. No test substance-related mortality or neurotoxicity occurred. The selected NOAEL is based on increased hepatic peroxisomal beta-oxidation, and the degeneration and/or disorganization of enamel organ ameloblast cells in males and females administered 100 mg/kg/d. In males additionally body weight decrements were observed which could be attributed to alterations to the teeth caused by fluorosis and therefore, decreased ability of animals to eat pelleted chow.