Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes
Remarks:
Hita Laboratory, Japan
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): ammonium perfluoro-3,6-dioxaoctanate (EEA-NH4)
- Physical state: white solid
- Analytical purity: 99.5%
- Lot/batch No.: RS4-56
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Inc. (Hino Breeding Center; 735; Shimokomatsuki, Hino-cho, Gamo-gun, Shiga 529-1633, Japan)
- Age at study initiation: 5 weeks
- Weight at study initiation: 132.7 - 168.9 g (males), 122.3 - 147.8 g (females)
- Fasting period before study: no
- Housing: hanging stainless steel cages with wire-mesh floor at 1 animal/cage
- Diet: MF pelleted diet (lot no. 050905 and 051101, Oriental Yeast), ad libitum
- Water: chlorinated water from the Hita City supply via automatic watering system with sipper tubes, ad libitum
- Acclimation period: 9 days (including 6 days quarantine)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 25
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Purified water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was accurately weighted and dissolved with purified water adding gradually to make 10.0 w/v% solution. The lower concentrations of 1.0, 0.25 and 0.05 w/v% were diluted from 10.0 w/v%. These were prepared once weekly and stored at a dark and cold place (refrigerator). The volume dosed was 10 mL/kg.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In the homogeneity test, top, middle and bottom layers of 0.05 and 10.0 w/v% formulations were taken (n=1) just after preparation, and quantitatively analysed using HPLC (n=1) after sample pretreatment. The samples were stored in a dark and cold place, and the middle layer of these was taken (n=1) after 5 and 7 days to be analysed. The formulations of 0.05 and 10.0 w/v% were stable for 7 days and showed good homogeneity.
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 5, 25, 100 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
five
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: 14-day oral repeated dose toxicity study
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: three times a day during the dosing period, once daily during the recovery period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: before dosing, and on days 1, 3, 8, 12, 17, 21, 16 and 28 during the dosing period; on days 1, 5, 10 and 14 during the recovery period

FOOD CONSUMPTION:
- Time schedule: before dosing, on days 3, 8, 15, 22 and 28 during the dosing period; on days 4, 8 and 14 during the recovery period

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at end of dosing period and at end of recovery period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all animals of the respective groups
- Parameters examined were red blood cell count (RBC), white blood cell count (WBC), hemoglobin concentration (Hb), hematocrit value (Ht), mean corposcular volume (MCV), mean corposcular hemoglobin (MCH), mean corposcular hemoglobin concentration (MCHC), platelet count, reticulocyte count, prothrombin time (PT), activated partial thromboplastin time (APTT), differentiation of leukocytes (neutrophils, eosinophils, basophils, lymphocytes, monocytes, large unstained cells (LUC), myeloblasts (myeloblasts (MyBl), promyeloblasts (PBl), myelocytes,metamyelocytes (My,Mt), Stab neutrophils,segmented neutrophils (N-Band,N-Seg), eosinophils, basophils, lymphocytes, plasmacytes, megakaryocytes, retoperitheliums, mastocytes, monocytes, proerythroblasts,basoerythroblasts (PEb,BEb), polychromatic erythroblasts,neuerythroblasts (PoEb,NEb), myeloblasts series/erythroblast series (M/E ratio).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at end of dosing period and at end of recovery period
- Animals fasted: Yes
- How many animals: all animals of the respective groups
- Parameters examined were aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), cholinesterase (ChE), γ-glutamyl transpeptidase (γ-GTP), total cholesterol (T-Cho), triglyceride (TG), glucose, total protein, albumin, A/G ratio, blood urea nitrogen (BUN), creatinine, total bilirubin (T-Bil), calcium, inorganic phosphorous (IP), sodium, potassium, chloride.

URINALYSIS: Yes
- Time schedule for collection of urine: at end of dosing period and at end of recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined were urine volume, colour, turbidity, urine specific gravity, pH, protein, glucose, occult blood, urinary sediments.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 4
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity, grip strength, motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The weights of the following organs were measured in all animals: liver, heart, kidneys, testes, epididymides, ovaries, brain, spleen thymus, adrenals. The relative organ weight was also calculated based on the body weight at the time of necropsy.
HISTOPATHOLOGY: Yes
Organs and tissues examined were trachea, lungs, incisors, stomach, intestine (duodenum to rectum, with Peyer's patches), liver, heart, kidneys, urinary bladder, testes, epididymides, prostate, seminal vesicle, ovaries, uterus, vagina, brain (cerebrum, cerebellum and pons), spinal cord, sciatic nerve, bone marrow (femur), axillar and mesenteric lymph nodes, spleen, thymus, pituitary gland, thyroids (with parathyroids), adrenals, eye ball.
Other examinations:
none
Statistics:
Data regarding body weights (excluding those at the time of necropsy) food intakes, hematological examinations, blood chemical examinations, urine volume and specific gravity, organ weights, grip strength and locomotor activity count were analyzed using the Bartlett's test for homogeneity of variance. If the variances were homogeneous at a significance level of 5%, one way analysis of variance was performed. If there was a significant difference in this analysis, the difference between the vehicle control group and each of the treatment groups was analyzed by the Dunnett's test.

If the variances were not homogeneous, the Kruskal-Wallis's test was used. If there was a significant difference in this analysis, the difference between the vehicle control group and each of the treatment group was analyzed by the nonparametric Dunnett's test.

Defecation and urination were analyzed using the Kruskal-Wallis's test. If there was a significant difference in this analysis, the difference between the vehicle control group and each of the treatment groups was analyzed by the nonparametric Duenna's test.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
No deaths occurred throughout the study. No abnormalities were observed in the clinical signs and detailed clinical observations during the dosing period and during the recovery period.

BODY WEIGHT
No abnormalities were noted in body weights.

FOOD CONSUMPTION
No abnormalities were noted during the dosing period. During the recovery period food intakes were increased in females in the 100 mg/kg bw group on day 4.

HAEMATOLOGY
In the 100 mg/kg group, at termination of the dosing period, decreased red blood cell count, hemoglobin concentrations and hematocrit values were observed in males and females. In the same group, increased reticulocyte count in females and prolonged prothrombin time in males were observed.
At termination of the recovery period, in the 100 mg/kg bw group, increased hemoglobin concentrations and hematocrit values were observed in males. In females in the 100 mg/kg bw recovery group no abnormalities were observed.

CLINICAL CHEMISTRY
In males of the 100 mg/kg bw group, at termination of the dosing period, increased alanine aminotransferase and A/G ratio, and decreased total cholesterol were observed. In females of the same group, total bilirubin was decreased.
In males of the 5 mg/kg bw group, at termination of the dosing period, total protein was decreased.
At termination of the recovery periodn, in males in the 100 mg/kg bw group, alanine aminotransferase was increased.

URINALYSIS
No abnormalities were noted in urinalysis.

NEUROBEHAVIOUR
No abnormalities were noted in sensorimotor function.

ORGAN WEIGHTS
At termination of the dosing period, absolute and relative kidney weights were increased in males of the 25 mg/kg bw group and higher. Absolute and relative liver weights were increased in males of the 100 mg/kg bw group. In females, no abnormalities were observed.
At termination of the recovery period, relative adrenal weight was increased in males in the 100 mg/kg bw group. In females in the same group, increased absolute kidney weight and decrease relative heart weight were observed.

GROSS PATHOLOGY
Elevation of limiting ridge in the forestomach in males and females and enlargement of the liver in males were observed in the 100 mg/kg bw group, at the end of the dosing period. No abnormalities were observed at the end of the recovery period.

HISTOPATHOLOGY: NON-NEOPLASTIC
After dosing period
In males round cell infiltration in the prostate and capsulitis of the spleen were observed in the vehicle control group. Hyperplasia of squamous epithelium in limiting ridge of the forestomach, diffuse hypertrophy of hepatocytes with granular degeneration in the liver, solitary cyst in medulla of the kidney, round cell infiltration in the prostate and capsulitis of the spleen were observed in the 100 mg/kg bw group.
In females mineralisation in corticomedullary junction and solitary cyst in medulla of the kidney were observed in the vehicle control group. Basophilic tubules and mineralisation in corticomedullary junction of the kidney were observed in the 5 mg/kg bw group. Basophilic tubules and mineralisation in corticomedullary junction of the kidney were observed in the 25 mg/kg bw group. Hyperplasia of squamous epithelium in limiting ridge of the forestomach, focal necrosis of hepatocytes in the liver, basophilic tubules and mineralization in corticomedullary junction of the kidney and hyperplasia of collecting tubular epithelium and transitional epithelium with interstitical cell infiltration in the unilateral kidney were observed in the 100 mg/kg bw group.

After recovery period
In males no abnormalities were observed.
In females basophilic tubules and mineralisation in corticomedullary junction of the kidney were observed in the vehicle control group. Mineralisation in corticomedullary junction of the kidney were observed in the 100 mg/kg bw group.

Effect levels

Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: increased absolute and relative kidney weights in males of the 25 mg/kg bw/day group and higher

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion