Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 August 2007 - 30 March 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study under GLP conditions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Colorless clear liquid
- Purity: 99.9%
- Lot/batch No.: 3SMZD
- Stability under test conditions: Stable

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan (Atsugi Farm)
- Age at study initiation: 9 weeks-old
- Weight at study initiation: Male: 317-361 g, Female: 199-251 g
- Fasting period before study: none
- Housing: Males and females were housed in stainless-steel wire-meshed cages except for gestation and lactation periods. During gestation and lactation periods, females were housed in polycarbonate cages.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: one week including quarantine period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.8-23.6°C
- Humidity (%): 49.8-61.8%
- Air changes (per hr): 6-20 times per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Dosing solutions were prepared daily before administration Using purified water as a vehicle, solution with a concentration of 30 mg/mL was prepared first. Then, the solution was diluted with vehicle and prepared as solutions with concentrations of 1 and 6 mg/L.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Because of high volatility of the test article, analytical verification of doses could not be performed.
Duration of treatment / exposure:
Male: 42 days including 14-days pre-mating period and mating period
Female: 42-52 days including 14 days pre-mating, mating and gestation periods, and days until lactation day 4.
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10, 60, 300 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
Administration groups:
Control and high dose groups: 7 males and 12 females per dose
Low and mid dose groups: 12 per sex per dose

Recovery groups:
Control and high dose groups: 5 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In 14-days preliminary study, a dose of 500 mg/kg/day tested was proved to be excess because of poor general conditions for the test animals. As a consequence, the top dose in the main study was set down to 300 mg/kg/day. The mid and low doses were set as 60 and 10 mg/kg/day, respectively.
- Rationale for selecting satellite groups: Satellite groups for recovery study were set for control and high dose groups.
- 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: 3 times (before and just after administration and about 3 hrs after administration) per day during administration period. Once daily during period other than administration.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: (Males) Day 1, 8, 15. 22, 29, 36, 42 and 43. (Females) Pre-mating Day 0, 7 and 17, once a week during mating period, gestation day (GD) 0, 7, 14 and 20, and lactation day 0 and 4. (Males and females, recovery groups) Day 50 and 56.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: (Males) Day 43; (Females) Lactation day 5; (Males and females, recovery) Day 57
- Anaesthetic used for blood collection: Yes (sodium pentobarbital)
- Animals fasted: Yes, 18-23 hrs from the day before.
- How many animals: 5 per sex per group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: the same schedule as haematology
- Animals fasted: Yes
- How many animals: 5 per sex per group
- Parameters checked in table 2 were examined.

URINALYSIS: Yes, but males only
- Time schedule for collection of urine: (Males) Day 40
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Parameters checked: pH, protein, glucose, keton body, birillubin, occult blood, urobilinogen

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: the day before start of administration, once a week during administration until Week-6.
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other: reactivity to stimuli

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
ORGAN WEIGHTS: Yes (see Table 3)
HISTOPATHOLOGY: Yes (see table 4)
Statistics:
For parametric data, homogeneity of variance was examined by Bartlett method. When the variance was equal, ANOVA was used. While variance was not equal or non-parametric data was applied, Kruskal-Wallis method was used. When significant difference was recognized among the groups, either Dunnett method or Dunnett multiple comparison test was used. For qualitative data, Dunnett multiple comparison test, Wilcoxon rank sum test, Fischer's exact probability, Student or Aspin-Welch's t-test were used as necessary.

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:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
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:
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 abnormality was found at 10 and 60 mg/kg groups of both sexes. At 300mg/kg, prone position and a decrease in locomotor activity were observed in both sexes. These changes were seen throughout the administration period in one to almost all animals from immediately after dosing and disappeared by 3 to 6 hours after dosing. One of the females died during the delivery following administration on day 24 of gestation. In this animal, prone position and a decrease in locomotor activity were observed and did not disappear even after 6 hours after dosing on the day before death.

BODY WEIGHT AND WEIGHT GAIN:
-There were no changes in body weight and body weight gain in male and female groups of 10 and 60 mg/kg. In females at 300 mg/kg, body weight decreased or tended to decrease from the late gestation until day 4 of lactation. The body weight of males in this group tended to decrease late in the administration period, but recovered during the recovery period.

FOOD CONSUMPTION:
-There were no changes in food consumption in male and female groups of 10 and 60 mg/kg. At 300 mg/kg, food consumption in the females showed a decrease or a tendency to decrease in the latter gestation and lactation period. Food consumption in males at 300 mg/kg was comparable to that of the control group during the administration period, but it decreased during the recovery period.

HAEMATOLOGY (Table 1):
-No change was noted up to 60 mg/kg in both sexes. At 300 mg/kg, reticulocyte count decreased in males, however, there was no difference in red blood cell count as compared to the control group. Decreased platelet count in males and decreased white blood cell count in females were noted. Since there were no histological changes and they recovered at the end of the recovery period, the effects on the organism were considered to be slight.


CLINICAL CHEMISTRY (Table 2):
-Increased triglyceride was noted in males and 1 female at 300 mg/kg. Although statistically not significant, a trend to increase in triglyceride was observed in male group at 60 mg/kg. Urea nitrogen increased in males at 300 mg/kg; however, creatinine did not differ from the control value without any effects on the renal function in other examinations. In female group at 300 mg/kg, decreases in calcium and potassium were observed. When compared with the background data of the test facility, potassium was within the range (mean±2S.D.) and calcium deviated from the range in 2 animals. Animal with the lowest value demonstrated reduced food consumption, therefore it was suspected to be a nutritional change. These changes were not seen at the end of the recovery period.

NEUROBEHAVIOUR:
-In the detailed clinical observation, functional tests and motor activity measurement, the following changes were observed as the clinical sign at 300 mg/kg; prone/lateral position, crawling position, and abnormal gait in both sexes, slow reactivity to stimuli and disappearance of aerial lighting reaction in some males, decreased grip strength in both sexes, decreased motor activity in both sexes. PHF is the main metabolite of inhalative anesthetic, sevoflurane and reported to have central nervous system depression action. Moreover, PHF is a monohydric alcohol (C3) and C1 to C13 alcohol is known to have anesthetic potential. Therefore, these neurobehavioral signs were considered to relate to the central nervous system depression and anesthetic action of PHF.

ORGAN WEIGHTS (Table 3):
-In males at 300 mg/kg, increase in relative liver weight, decrease in absolute and relative adrenal weight were observed. Decrease in adrenal weight was also observed in males at 10 and 60 mg/kg. When compared with the background data of the test facility, low values in groups of 10 and 60 mg/kg were within the range (mean±2S.D.). Therefore, these were considered not toxicologically significant.

GROSS PATHOLOGY:
-In scheduled-necropsied animals, no treatment-related changes were observed.
-In one female dead animal, small size of spleen and thymus as well as dilatation of renal pelvis were observed. Moreover, black patch in mucosa of fundic gland or pylric region.

HISTOPATHOLOGY: NON-NEOPLASTIC (Table 4)
-Changes considered as treatment-related were limited to both sexes groups at 300 mg/kg.
-In scheduled-necropsied animals, centrilobular hypertrophy of hepatocytes were observed in 2 males and 3 females at this dose. In the same male group, erosion of duodenal mucosa was observed. In the recovery group, theses changes were not recognized.
-In dead female animal, the following changes were observed: erosion with hemorrhage in the fundic mucosa and erosion of the duodenal mucosa with hypertrophy of the duodenal glands, erosion of cecum, hypertrophy of the cortical cells in the fascicular zone, atrophy of the hematopoietic tissues, and necrosis of lymphocytes in thymus and mandibular lymph node.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: 300 mg/kg: death (female), clinical signs (depression of central nervous system, anesthetic action)(both sexes), decrease in body weight (both sexes), irritating changes in gastrointestinal tract.
Dose descriptor:
NOEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: 60 mg/kg: increase in triglyceride
Dose descriptor:
NOEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: 60 mg/kg: No abnormality

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1 Hematological examinations in rats treated orally with PHF in the combined repeated dose and reproductive/developmental toxicity screening test.

Sex Dose level (mg/kg) Administration period Recovery period
0 10 60 300 0 300
Male Number of animals 5 5 5 5 5 5
RBC (X106/μL) 9.044±0.321 8.924±0.415 8.892±0.325 9.294±0.211 9.184±0.234 9.306±0.285
Hemoglobin (g/dL) 16.30±0.72 16.56±0.71 16.30±0.57 16.20±0.59 16.42±0.68 16.14±0.41
Hematocrit (%) 46.12±1.88 46.84±1.98 45.86±1.71 46.10±1.66 46.18±1.57 45.52±1.39
Reticulocyte (%) 3.680±0.385 3.246±0.513 3,218±0.199 2.358±0.658** 3.280±0.153 2.988±0.300
MCV (fL) 51.02±2.02 52.50±1.65 51.58±1.87 49.64±2.57 50.28±1.42 48.94±1.75
MCH (pg) 18.04±0.65 18.56±0.51 18.32±0.36 17.46±0.91 17.88±0.46 17.36±0.51
MCHC (g/dL) 35.36±0.30 35.38±0.42 35.56±0.67 35.14±0.42 35.58±0.48 35.44±0.32
Platelet (X103/μL) 1217.8±79.6 1232.2±108.9 1168.4±120.4 905.0±83.4** 1250.4±127.2 1116.6±116.8
PT (sec) 21.46±3.58 23.52±5.15 23.36±5.48 22.40±4.48 22.82±3.97 27.84±4.28
APTT (sec) 20.72±2.74 22.14±2.43 19.78±2.34 18.52±2.20 20.98±2.22 22.58±1.00
WBC (X103/μL) 10.128±2.781 10.294±2.486 10.028±2.751 8.634±2.324 9.704±2.438 7.482±3.276
Lymphocyte (%) 74.64±6.30 83.04±3.68 74.70±4.48 78.40±5.25 70.86±6.77 78.22±5.12
Neutrophil (%) 21.14±5.91 14.02±3.14 21.50±4.10 16.64±3.99 25.34±6.75 17.12±5.31
Eosinophil (%) 1.04±0.19 1.00±0.51 1.28±0.46 1.30±0.69 1.42±0.58 1.70±0.76
Basophil (%) 0.00±0.00 0.02±0.04 0.00±0.00 0.00±0.00 0.02±0.04 0.00±0.00
Monocyte (%) 3.18±0.79 1.92±0.69 2.52±0.65 3.66±1.60 2.36±0.19 2.96±0.61
Female Number of animals 5 5 5 5 5 5
RBC (X106/μL) 7.296±0.423 7.304±0.305 7.582±0.509 7.552±0.725 8.232±0.486 8.942±0.461*
Hemoglobin (g/dL) 14.10±0.81 14.10±0.51 14.46±0.70 14.10±0.85 15.60±0.60 15.80±0.69
Hematocrit (%) 41.22±2.26 41.20±1.05 41.78±1.48 42.14±2.17 44.28±2.05 44.74±1.95
Reticulocyte (%) 8.622±1.222 8.624±3.069 6.446±1.999 9.446±4.226 2.910±0.984 2.308±0.895
MCV (fL) 56.50±1.21 56.46±1.05 55.20±1.94 56.02±3.10 53.82±1.40 50.08±1.84**
MCH (pg) 19.32±0.48 19.28±0.40 19.10±0.58 18.72±0.92 18.98±0.57 17.68±0.43**
MCHC (g/dL) 34.20±0.28 34.20±0.62 34.60±0.65 33.48±0.59 35.26±0.30 35.30±0.51
Platelet (X103/μL) 1296.4±97.0 1302.2±265.2 1122.2±127.6 1108.6±122.3 1024.0±129.9 1115.8±192.4
PT (sec) 18.90±0.68 18.40±0.63 18.06±0.76 18.20±0.65 16.32±0.85 15.90±0.48
APTT (sec) 15.82±0.76 14.86±1.68 14.92±0.93 14.22±0.97 14.92±0.54 15.74±1.36
WBC (X103/μL) 10.938±2.945 11.948±2.305 8.986±1.327 6.266±1.350** 5.230±1.957 4.804±1.385
Lymphocyte (%) 67.68±4.45 68.86±6.85 64.56±7.28 72.34±9.56 80.46±3.17 77.22±8.51
Neutrophil (%) 28.04±4.30 26.98±7.73 29.88±6.94 21.42±9.83 15.16±2.92 19.02±8.11
Eosinophil (%) 1.04±0.40 0.66±0.21 0.98±0.41 1.00±0.32 1.48±0.49 1.38±0.28
Basophil (%) 0.00±0.00 0.00±0.00 0.02±0.04 0.00±0.00 0.00±0.00 0.00±0.00
Monocyte (%) 3.24±0.57 3.50±1.13 4.56±1.60 5.24±1.45 2.90±1.19 2.38±0.52

Values are expressed as Mean ± S.D.

Significantly different from 0 mg/kg group; * p<0.05, ** p<0.01

Table 2 Blood chemical examination in rats treated orally with PHF in the combined repeated dose and reproductive/developmental toxicity screening test

Sex Dose level (mg/kg) Administration period Recovery period
0 10 60 300 0 300
Male Number of animals 5 4 5 5 5 5
ASAT (U/L) 89.6±15.1 92.3±9.7 127.4±27.5** 102.8±8.0 106.0±19.8 81.8±14.9
ALAT (U/L) 26.6±1.8 26.0±7.4 27.0±6.0 31.0±4.6 36.0±12.9 25.8±1.6
gamma-GT (U/L) 0.6±0.5 1.0±0.0 1.0±0.0 1.0±0.0 0.6±0.5 0.4±0.5
ALP (U/L) 420.0±76.5 399.0±159.1 471.0±74.6 564.4±74.7 304.2±45.1 335.6±24.0
Total bilirubin (mg/dL) 0.00±0.00 0.00±0.00 0.00±0.00 0.02±0.04 0.00±0.00 0.00±0.00
Urea nitrogen (mg/dL) 14.40±1.50 14.20±1.06 14.68±0.67 17.54±1.34** 13.98±1.53 12.66±1.88
Creatinine (mg/dL) 0.28±0.04 0.28±0.05 0.30±0.00 0.32±0.04 0.28±0.04 0.28±0.04
Glucose (mg/dL) 125.4±6.3 117.0±9.5 128.2±9.1 134.6±9.7 147.8±10.7 154.6±13.1
Total chol. (mg/dL) 58.4±15.8 60.0±14.7 50.0±9.2 62.4±14.7 66.6±7.6 68.8±16.0
Triglyceride (mg/dL) 20.4±6.9 13.8±3.6 36.8±20.8 96.2±46.6* 29.4±13.0 33.0±11.0
Total protein (g/dL) 6.80±0.16 6.90±0.22 6.84±0.44 6.98±0.22 7.18±0.19 7.26±0.15
Albumin (g/dL) 2.94±0.09 2.98±0.21 2.90±0.16 3.06±0.05 3.02±0.11 2.96±0.05
A/G ratio 0.750±0.049 0.758±0.046 0.774±0.032 0.790±0.032 0.730±0.047 0.694±0.022
Calcium (mg/dL) 9.96±0.40 9.88±0.29 9.76±0.34 10.06±0.28 9.98±0.27 9.86±0.05
Inorganic phos. (mg/dL) 8.22±0.46 8.03±0.47 7.68±0.29 8.22±0.27 7.04±0.34 7.20±0.62
Na (mmol/L) 150.0±1.2 150.0±0.8 149.8±1.3 150.6±1.1 148.0±0.7 147.6±0.9
K (mmol/L) 4.46±0.36 4.70±0.22 4.52±0.19 4.30±0.07 4.28±0.36 4.38±0.13
Cl (mmol/L) 104.4±1.1 105.5±1.3 105.8±1.5 105.8±2.2 104.4±1.3 104.4±0.5
Female Number of animals 5 5 5 5 5 5
ASAT (U/L) 133.2±36.8 113.8±15.6 109.8±12.8 107.6±11.5 128.6±97.4 99.2±12.8
ALAT (U/L) 57.6±16.3 47.8±11.0 53.4±7.6 45.8±11.4 50.2±63.3 24.8±5.3
gamma-GT (U/L) 1.0±0.0 1.0±0.0 1.0±0.0 1.0±0.0 0.8±0.4 0.4±0.5
ALP (U/L) 257.4±35.0 267.8±82.5 306.8±178.3 255.0±59.8 145.6±28.7 140.6±23.8
Total bilirubin (mg/dL) 0.00±0.00 0.00±0.00 0.00±0.00 0.02±0.04 0.06±0.05 0.02±0.04
Urea nitrogen (mg/dL) 17.82±2.07 21.54±2.25 20.44±5.50 21.56±2.87 15.50±1.30 13.66±1.11*
Creatinine (mg/dL) 0.34±0.05 0.34±0.05 0.34±0.05 0.34±0.05 0.36±0.05 0.34±0.05
Glucose (mg/dL) 121.8±19.3 119.8±8.7 129.2±11.4 123.4±19.2 136.2±36.5 135.4±16.5
Total chol. (mg/dL) 68.0±10.3 68.6±9.1 86.2±14.8* 62.6±6.3 89.0±16.4 79.8±14.6
Triglyceride (mg/dL) 37.2±10.1 44.8±18.3 41.8±10.6 62.6±58.3 13.0±6.0 11.0±3.2
Total protein (g/dL) 7.14±0.11 7.40±0.41 7.22±0.42 6.62±0.50 7.82±0.36 7.44±0.30
Albumin (g/dL) 3.10±0.07 3.30±0.07 3.12±0.23 2.92±0.22 3.60±0.29 3.32±0.13
A/G ratio 0.764±0.036 0.798±0.072 0.752±0.041 0.790±0.054 0.842±0.072 0.804±0.026
Calcium (mg/dL) 11.16±0.35 11.42±0.43 11.12±0.41 10.32±0.76* 10.16±0.34 10.20±0.22
Inorganic phos. (mg/dL) 9.58±0.98 9.92±0.78 9.54±0.66 9.98±1.34 6.46±0.33 6.75±0.71
Na (mmol/L) 147.6±1.5 147.2±1.3 147.4±2.3 146.0±1.0 146.4±1.1 148.0±0.8
K (mmol/L) 4.86±0.18 4.76±0.29 4.58±0.15 4.30±0.32** 4.26±0.19 4.28±0.15
Cl (mmol/L) 105.0±1.7 103.4±2.9 103.4±2.3 104.0±3.4 105.8±1.5 107.5±1.3

Values are expressed as Mean± S.D.

Significantly different from 0 mg/kg group; * p<0.05, ** p<0.01

Table 3 Absolute and relative organ weights in rats treated orally with PHF in the combined repeated dose and reproductive/developmental toxicity screening test

Sex Dose level (mg/kg) Administration period Recovery period
0 10 60 300 0 300
Male Number of animals 5 5 5 5 5 5
Final body weight (g) 452.3±29.6 (7)a) 474.7±33.2 (12) 471.6±30.7 (12) 429.7±39.5 (7) 515.0±28.8 477.2±22.3*
Absolute organ weight
Brain (g) 2.100±0.073 2.138±0.042 2.092±0.084 1.976±0.042* 2.172±0.073 2.074±0.005*
Thymus (mg) 386.6±121.9 289.4±63.2 425.0±81.1 250.4±38.6* 358.4±58.8 341.4±96.8
Heart (g) 1.572±0.112 1.554±0.216 1.584±0.163 1.506±0.188 1.680±0.067 1.764±0.091
Liver (g) 11.570±0.871 11.522±1.508 12.574±0.977 12.246±1.757 13.540±1.032 13.436±1.162
Spleen (g) 0.830±0.029 0.838±0.171 0.756±0.085 0.686±0.106 0.872±0.096 0.756±0.056*
Kidneys (g) 2.968±0.212 3.092±0.381 3.134±0.259 2.546±0.241 3.262±0.121 3.100±0.139
Adrenals (mg) 80.80±13.00 69.06±6.50 63.54±9.68* 48.02±6.57** 75.24±8.77 66.66±10.30
Testes (g) 3.493±0.152 (7) 3.422±0.300 (12) 3.298±0.400 (12) 3.233±0.381 (7) 3.350±0.100  3.494±0.128
Epididymides (g) 1.389±0.088 (7) 1.397±0.113 (12) 1.354±0.175 (12) 1.263±0.173 (7) 1.480±0.129 1.506±0.077
Relative organ weight 
Brain (g%) 0.470±0.022 0.458±0.038 0.426±0.019 0.476±0.044 0.422±0.028 0.436±0.018
Thymus (mg%) 86.04±24.93 61.14±9.92 86.82±16.29 59.68±7.44 69.48±10.22 71.38±19.73
Heart (g%) 0.352±0.029 0.330±0.024 0.324±0.038 0.360±0.027 0.328±0.008 0.372±0.032*
Liver (g%) 2.592±0.195 2.440±0.139 2.566±0.149 2.914±0.187* 2.628±0.153 2.812±0.114
Spleen (g%) 0.186±0.013 0.176±0.021 0.152±0.013** 0.164±0.009 0.170±0.010 0.158±0.018
Kidneys (g%) 0.664±0.039 0.656±0.052 0.642±0.056 0.608±0.019 0.634±0.025 0.648±0.026
Adrenals (mg%) 18.02±2.34 14.78±2.10* 12.92±1.38** 11.48±1.53** 14.62±1.68 13.98±2.02
Testes (g%) 0.774±0.065 (7) 0.723±0.073 (12) 0.699±0.063 (12) 0.754±0.081 (7) 0.654±0.052 0.732±0.022*
Epididymides (g%) 0.309±0.036 (7) 0.293±0.021 (12) 0.288±0.034 (12) 0.296±0.048 (7) 0.288±0.033 0.316±0.018
Female Number of animals 5 5 5 5 5 5
Final body weight (g) 307.6±19.4 305.4±39.9 329.4±40.7 277.4±10.8 311.0±71.4 285.0±24.0
Absolute organ weight
Brain (g) 1.916±0.103 1.950±0.091 2.022±0.067 1.920±0.037 1.924±0.053 1.866±0.100
Thymus (mg) 288.6±94.5 354.0±156.3 279.4±49.9 216.6±57.4 348.6±103.8 286.4±50.4
Heart (g) 1.088±0.099 1.048±0.168 1.078±0.100 1.028±0.052 0.990±0.181 0.960±0.088
Liver (g) 10.374±0.788 10.740±1.407 11.190±0.742 9.342±0.528 8.426±2.185 6.768±0.465
Spleen (g) 0.720±0.095 0.808±0.178 0.706±0.091 0.656±0.112 0.554±0.107 0.484±0.031
Kidneys (g) 2.104±0.160 2.052±0.248 2.106±0.100 1.940±0.108 1.956±0.358 1.700±0.110
Adrenals (mg) 83.78±9.67 75.28±10.30 79.60±4.13 70.86±7.37 77.92±13.54 71.10±11.89
Relative organ weight 
Brain (g%) 0.624±0.027 0.650±0.083 0.620±0.068 0.692±0.019 0.644±0.125 0.656±0.041
Thymus (mg%) 93.56±28.74 112.54±36.35 84.62±8.28 77.66±17.98 110.80±7.34 100.50±15.17
Heart (g%) 0.354±0.026 0.346±0.044 0.326±0.013 0.374±0.025 0.320±0.024 0.334±0.011
Liver (g%) 3.370±0.098 3.518±0.102 3.422±0.330 3.374±0.249 2.696±0.118 2.378±0.039**
Spleen (g%) 0.234±0.018 0.266±0.041 0.216±0.044 0.238±0.036 0.178±0.015 0.172±0.019
Kidneys (g%) 0.684±0.017 0.674±0.058 0.648±0.085 0.698±0.058 0.634±0.043 0.598±0.027
Adrenals (mg%) 27.36±3.88 24.90±3.93 24.42±2.78 25.58±2.76 25.32±1.60 24.94±3.58

a) Number of animals examined.

Values are expressed as Mean± S.D.

Significantly different from 0 mg/kg group; * p<0.05, ** p<0.01

Applicant's summary and conclusion

Conclusions:
NOAEL for the repeated dose toxicity of PHF was judged to be 60 mg/kg/day for males and females, since a death in females, clinical signs related to central nervous system depression and anesthetic changes, decreased body weight, and irritative changes in the gastrointestinal tract in both sexes were observed in the 300 mg/kg group. The NOEL was judged to be 10 mg/kg/day for males and 60 mg/kg/day for females, since increased triglyceride was observed in males without any organic alterations and no test substance-related abnormalities were observed in females at 60 mg/kg.
Executive summary:

In accordance with the OECD guideline on combined repeated dose and reproductive/developmental toxicity screening test, 2-propanol, 1,1,1,3,3,3-hexafluoro- (PHF) was studied for oral toxicity in rats at doses of 0, 10, 60, and 300 mg/kg/day.

 

Prone position and a decrease in locomotor activity were observed in both sexes at 300 mg/kg. These changes were seen throughout the administration period in one to almost all animals from immediately after dosing and disappeared by 3 to 6 hours after dosing. One of the females died during the delivery following administration on day 24 of gestation. In this animal, prone position and a decrease in locomotor activity were observed and did not disappear even after 6 hours after dosing on the day before death. These changes were observed until the dosing on the day of death. PHF is the main metabolism of inhalation anesthetic, sevoflurane and reported to have central nervous system depression action.

Moreover, PHF is a monohydric alcohol (C3) and C1to C13 alcohol is known to have anesthetic potential. Based on this, prone position and a decrease in locomotor activity observed after administration of PHF were considered to be attributable to the central nervous system depression and anesthetic action. In the dead female, it was considered that these actions occurred strongly during delivery, and consequently the condition of the animal deteriorated and was followed by death. In the detailed clinical observation, functional tests and motor activity measurement, the following changes related to the central nervous system depression and anesthetic action were observed as the clinical sign at 300 mg/kg; prone/lateral position, crawling position, and abnormal gait in both sexes, slow reactivity to stimuli and disappearance of aerial lighting reaction in some males, decreased grip strength in both sexes, decreased motor activity in both sexes. In females at 300 mg/kg, body weight and food consumption decreased or tended to decrease in the late of gestation until day 4 of lactation.

The body weight of males in this group tended to decrease late in the administration period, but recovered during the recovery period. In addition, food consumption was comparable to that of the control group during the administration period, but it decreased during the recovery period. In the 300 mg/kg group, 1 male showed erosion of the duodenal mucosa and the dead animal showed the erosion with hemorrhage in the fundic mucosa and erosion of the duodenal mucosa with hypertrophy of the duodenal glands. In a dose-range finding study, reddish patch in the glandular stomach mucosa, thickening of the wall of fore- or glandular stomach were noted in the 500 mg/kg group. According to the material safety data sheet, PHF is categorized into corrosive substance, which irritates skin, eyes and mucosa. Therefore, erosion in the stomach and duodenal mucosa was considered to be caused by irritability of the test substance. In addition, it is reported that the duodenal gland acts on the mucosal protection and hypertrophy of the duodenal glands is often seen as the regenerative change to intermittent hemorrhage at the upper gastrointestinal tract and erosion/ulcer of the duodenal mucosa. As for dead animal, hypertrophy of the cortical cell in the fascicular zone, atrophy of the hematopoietic tissues, and necrosis of the lymphocyte were considered to be stress-related secondary changes attributed to erosion of the gastric and duodenal mucosa. Erosion of the cecum mucosa was suspected to be induced by the hypofunction of mucosal protection accompanied by atrophy of the hematopoietic tissues and lymphocytic necrosis. In males at 300 mg/kg, decreased thymus weight without any organic alteration was considered to be stress-related. Increased triglyceride was noted in males and 1 female at 300 mg/kg. This change was also noted in males at 60 mg/kg. PHF is known to be a MCD (malonyl-CoA decarboxylase) inhibitor. It is reported the malonyl CoA inhibits the activity of CPT-1 (carnitine palmitoyltransferase I) and suppresses breakdown of fatty acid. On the other hand, hypertrophy of the centrilobular hepatocytes was observed in both sexes and liver weight increased in males at 300 mg/kg. Since there were no fatty degeneration or any increases in liver enzyme indicating liver damage, and it was a reversible change, it was suspected to be an adoptive change. From these results, possibility of effects of PHF and MCD inhibition on fatty metabolism were not clarified. As for increased triglyceride, since it was reversible and not accompanied by any histological changes, it was not considered to be significant.

Although reticulocyte count decreased in males at 300 mg/kg, there was no difference in red blood cell count as compared to the control group. Decreased platelet count in males and decreased white blood cell count in females were noted. Since there were no histological changes and they recovered at the end of the recovery period, the effects on the organism were considered to be slight. Urea nitrogen increased in males at 300 mg/kg; however, creatinine did not differ from the control value without any effects on the renal function in other examinations. Calcium and potassium decreased in females. When compared with the background data of the test facility, potassium was within the range (mean±2S.D.) and calcium deviated from the range in 2 animals. Animal with the lowest value demonstrated reduced food consumption, therefore it was suspected to be a nutritional change. These changes were not seen at the end of the recovery period. Decreased adrenal weight was noted in males at 300 mg/kg; however, it recovered during the recovery period. There was no histological change and its toxicological significance remained unclear.

There were no test substance related changes in the urinalysis of males.

 

From the above results, NOAEL for the repeated dose toxicity of PHF was judged to be 60 mg/kg/day for males and females, since a death in females, clinical signs related to central nervous system depression and anesthetic changes, decreased body weight, and irritative changes in the gastrointestinal tract in both sexes were observed in the 300 mg/kg group. The NOEL was judged to be 10 mg/kg/day for males and 60 mg/kg/day for females, since increased triglyceride was observed in males without any organic alterations and no test substance-related abnormalities were observed in females at 60 mg/kg.