Registration Dossier

Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 Aug 2007 - 30 Mar 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 Aug 2007 - 30 Mar 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
March 22, 1996
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
yes
Remarks:
Pups were terminated on postnatal day 4, the T3, T4, TSH levels were not measured, anogenital distance was ot measured, nipple retention was not recorded.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: sealed in bottle in a fridge
- Stability under test conditions: verified by IR at the beginning and at the end of the study
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc., Yokohama, Japan
- Age at study initiation: 9-weeks old
- Weight at study initiation: 317 - 361 g (male), 199 - 251 g (female)
- Fasting period before study: none
- Housing: 1 male and 1 female was housed in one stainless-steel wire-meshed cages except for gestation and lactation periods. During gestation and lactation periods, one female was housed in a polycarbonate cage.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: one week including quarantine period

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

IN-LIFE DATES: From 12 Sep 2007 To 13 Nov 2007
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, a 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/mL.
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 postpartum until lactation day 4.
Frequency of treatment:
7 days/week
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
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/Satellite groups:
Control and high dose groups: 5 per sex per dose
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: In a 14-day preliminary study, a dose of 500 mg/kg bw/day was considered too high for the main study because of poor general conditions for the test animals. As a consequence, the top dose in the main study was set to 300 mg/kg bw/day. The mid- and low doses were set as 60 and 10 mg/kg bw/day, respectively.
- Rationale for selecting satellite groups: Satellite groups for recovery study were set for control and high dose groups to assess the recovery from any observed effects.
- Post-exposure recovery period in satellite groups: 14 days
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 3 times (before and just after administration and about 3 h after administration) per day during administration period. Once daily during period recovery period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: the first observation after the first administration, thereafter weekly. Detailed clinical observations were recorded after administration, however, no information how long after dosing is available.

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

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

FOOD EFFICIENCY: No
- 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, satellite groups: Day 57.
- Anaesthetic used for blood collection: Yes (sodium pentobarbital)
- Animals fasted: Yes, 18 - 23 h
- How many animals: 5 per sex per group
- Parameters checked: RBC, hemoglobin, hematocrit, reticulocyte, mean cell volume (MCV), mean cell hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet, prothrombin time (PT), activated partial thromboplastin time (APTT), white blood cell (WBC), lymphocyte, neutrophil, eosinophil, basophil, and monocyte.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: the same schedule as haematology
- Anaesthetic used for blood collection: Yes (sodium pentobarbital)
- Animals fasted: Yes, 18 - 23 h
- How many animals: 5 per sex per group
- Parameters checked: aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), gamma-glutamyl transferase (GT), alkaline phosphatase (ALP), toal bilirubin, urea nitrogen, creatinine, glucose, total cholesterol, triglyceride, total protein, albumin, albumin to globulin (A/G) ratio, calcium, inorganic phosphate, sodium, potassium, and chloride.

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, and urobilinogen

IMMUNOLOGY: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: the day before start of administration, and once a week during administration until Week 6.
- Dose groups that were examined: all groups
- Battery of functions tested: sensory activity, grip strength, motor activity, and reactivity to stimuli.
Sacrifice and pathology:
SACRIFICE:
Male animals: Rats were euthanized by exsanguination under anesthesia on the day after the last administration.
Maternal animals: Rats were euthanized by exsanguination under anesthesia on Day 4 of lactation.

GROSS NECROPSY: Yes, Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

GROSS PATHOLOGY: Yes; spleen, liver, heart, lymph node, mandibular, lymph node, mesenteric, thymus, bone marrow, femur, trachea, lung (and bronchus), stomach, small intestine, duodenum, small intestine, jejunum, small intestine, ileum, large intestine, cecum, colon, rectum, kidney, urinary bladder, ovary, uterus, vagina, mammary gland, pituitary, thyroid, parathyroid, adrenal, brain, spinal cord, and sciatic nerve.

ORGAN WEIGHTS: Yes; brain, heart, liver, kidney, adrenal gland, thymus, spleen, testis and epididymis.

HISTOPATHOLOGY: Yes
Control and 300 mg/kg bw/day groups: brain, pituitary gland, thymus, lymph node, trachea, lung, stomach, intestine (duodenum, jejunum, ileum, cecum, colon, rectum), thyroid gland, parathyroid (bilateral), heart, liver , spleen, kidney (bilateral), adrenal (bilateral), bladder, testis (bilateral), epididymis (bilateral), seminal vesicle (including coagulation gland), prostate abdominal lobe, ovary (bilateral), uterus, sputum, bone marrow of right femur, right sciatic nerve, spinal cord, and gross abnormal area.
All groups: liver, small intestine(duodenum), testes, and epididymides.
The above listed organs and tissues of all animals were collected, fixed with 10 vol% neutral phosphate buffered formalin, and stored. Male testis and epididymis were fixed with bouin solution and stored in 10 vol% neutral phosphate buffered formalin solution.
Statistics:
For parametric data, homogeneity of variance was examined by Bartlett method. When the variance was equal, ANOVA was used. When 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.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No abnormality was found in the 10 and 60 mg/kg bw/day groups of both sexes. In the 300 mg/kg bw/day group, prone position and a decrease in locomotor activity were observed in both sexes. These changes were seen throughout the administration period in 1/12 to 9/12 animals from immediately after dosing and disappeared by 3 to 6 hours after dosing. No clinical signs were noted during the recovery period. The effects were considered to be treatment-related and toxiclogically relevant.
Mortality:
mortality observed, treatment-related
Description (incidence):
One female in the 300 mg/kg bw/day group died during 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 changes:
effects observed, treatment-related
Description (incidence and severity):
There were no changes in body weight and body weight gain in male and female groups of 10 and 60 mg/kg bw/day compared with control. In females at 300 mg/kg bw/day, body weight gain showed 10% decrease from the late gestation until day 4 of lactation compared with control (see Table 2 under “Any other information on results including tables”). This change was statistically significant on lactation day 4. Body weight gains of males in the 300 mg/kg bw/day group showed almost 9% decrease compared with control after Day 22 (see Table 1 under “Any other information on results including tables”). This change was significant on Day 50 and 56. Although the body weight remained significant low during the recovery period, the weight gain during the last week of the recovery males was similar to that in the control group. The females in the recovery group did not gain weight from Day 42 to 56. The effects on body weight are treatment-related and toxicologically relevant, adverse effect.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There were no changes in food consumption in male and female groups of 10 and 60 mg/kg bw/day compared with control. Food consumption in males at 300 mg/kg bw/day was similar to that of the control group during the administration period. The food consumption was significantly reduced (by 20%) during the recovery period on Day 50 and Day 54 in males at 300 mg/kg bw/day group compared with the control. See Table 3 under “Any other information on results including tables”. The food consumption in the females showed a 20% decrease on Gestation Day 20 and 35% decrease on Lactation Day 4 at 300 mg/kg bw/day group compared with the control. See Table 4 under “Any other information on results including tables”. In the recovery group, the reduced food consumption was significant in females in the 300 mg/kg bw/day group on Day 56, during the recovery period. The effects are treatment-related and toxicologically relevant, adverse effect.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No change was noted at 10 and 60 mg/kg bw/day in both sexes compared with control. At 300 mg/kg bw/day, 35% decreased reticulocyte count and 25% decreased platelet count was noted in males at the end of the administration compared with control. The effects were not considered to be toxicologically relevant, since there were no histological changes and the changes were only observed in the males. A 40% decreased white blood cell count in females at 300 mg/kg bw/day was noted at the end of the administration compared with control. The effects were not considered to be toxicologically relevant, since there were no histological changes and the changes were only observed in the females. At the end of the recovery period, 8% increased red blood cell and 7% decresased MCV and 7% decresased MCH was noted in females of the 300 mg/kg bw/day group compared with control. See Table 5 under “Any other information on results including tables”. These changes were also not considered to be toxicologically relevant since the changes were not observed at the end of the administration period.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A 450% increased triglyceride level was noted in the male 300 mg/kg bw/day group compared with control. Although statistically not significant, 180% and 160% increase in triglyceride was observed in the male 60 mg/kg bw/day and 300 mg/kg bw/day group compared with control, respectively. For the triglycerides, this may be related to the test substance causing increased metabolic activity in the liver, also seen as an increase in liver weight in the high-dose males, and therefore treatment-related. However it is reversible and therefore considered to be an adaptive response. Urea nitrogen showed 18% increase in males at 300 mg/kg bw/day compared with control; however, creatinine did not differ from the control value without any effects on the renal function in other examinations. This change was not considered as treatment-related effect. In female group at 300 mg/kg bw/day, decreases in calcium (8% decrease) and potassium (12% decrease) were observed in comparison with control. The potassium values was within ± 2 S.D of the historical control range. This change was considered as an incidental change. With regard to calcium, the change was minimal and was not observed at the end of the recovery period. Therefore, the changes in calcium level is considered to be incidental.
The ASAT values showed 15 – 20% decrease in females in all administration groups compared with control. When compared with the historical control range of the test facility, two animals of the control group had higher values (mean + 2 S.D.). Furthermore the resultswere non-significant, and this change was not considered a treatment-related effect. Males in the 60 mg/kg bw/day group showed 40% increased ASAT value compared with control. This change was not considered as the treatment-related effect since there was no dose-depending manner noted. Females in the 60 mg/kg bw/day group showed increased total cholesterol compared with control. However, this change was not considered as the treatment-related effect since a dose-depended increase was not observed.
At the end of recovery period, the satellite females in the 300 mg/kg bw/day group showed non-significant changes in ASAT (30% decrease), and ALAT (200% decrease) compared with control. However, the same change was not observed at the end of administration period, and as a result of comparison with the background data of the test facility, all cases were within the range of the mean ± 2 S.D. of the background data. It was considered as an incidental change. See table 6 under “Any other information on results including tables”.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
No change was noted up to 60 mg/kg bw/day in both sexes compared with control. In the 300 mg/kg bw/day groups, prone position (1 – 6 males in 12 males between week 3 and 6, 1 – 3 females in 17 females between week 1 and 3, 5 and 6), lateral position (2/12 males at week 3, 1/17 female at week 5), crawling position (1 – 3/12 males at week 2 and 4, 1 – 3/17 females at week 1, 2, 3, 5 and 6) and abnormal gait in both sexes (1 – 10/12 males during week 2 and 6, 1 – 6/17 females during week 1 and 6) were recorded.
As for the functional test, males in the 300 mg/kg bw/day group (within 5 males) showed abnormal reactivity to stimuli (2 male without reaction to approach response and touch response, 1 male without reaction to auditory reponse, 1 male with slight bad aerial righting reaction, 2 males with disapperance of aerial righting reaction).

In the 300 mg/kg bw/day group, grip strength was decreased in males (38% for forelimb) and females (36% for forelimb and 36% for hindlimb). Motor activity after the administration between 10 and 50 minutes was sinigifanctly decreased (90% decrease in males and 98% decrease in female).

These effects on behaviour and functional parameters were not observed during the recovery period, indicating they were reversible changes over time. Aseffects were serious, and consistently observed in mutiple parameters in both sexes from 2-3 weeks after administration started, In the high-dose group, these are considered to be treatment-related, toxicologically relevant adverse effects.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In males at 300 mg/kg bw/day, increase in relative liver weight (12% compared with control) was observed. This change was considered as a treatment-related adverse effect. The decrease in absolute (41%) and relative (37%) adrenal weight were observed in the males 300 mg/kg bw/day group compared with control. This was considered to be stress-induced, as this is a know effect in the species. This change was also observed in males at 10 (14% decrease in the absolute and 18% decrease in the relative weight) and 60 mg/kg bw/day (21% decrease in absolute weight and 28% decrease in relative weight) compared with control. When compared with the historical control range of the test facility, low values in groups of 10 and 60 mg/kg bw/day were within the range (mean ± 2 S.D.). Therefore, these were considered not toxicologically significant. In females no changes in the liver and adrenals were observed during the administration period. At the end of the recovery period, decreased liver relative weight was noted. This change was not considered as treatment-related effect since no change was observed at the end of the administration period.
The decreased absolute brain weight was noted at the end of administration period in the male 300 mg/kg bw/day group compared with control. The decreased final body weight, the decreased absolute brain weight, the decreased absolute spleen weight, the increased relative heart weight, and increased relative testes weight were observed in the male 300 mg/kg bw/day at the end of recovery period compared with control. There changes were regarded as incidental effects due to a lack of correlation with histopathological observations and the limited changes in %.
See Table 7 under “Any other information on results including tables”.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In scheduled-necropsied animals, no treatment-related changes were observed.
In one female animal that died on Day 24 of gestation, a small size of spleen and thymus were observed. Moreover, black patch in mucosa of fundic gland or pylric region were observed, which may be due to the corrosive effect of the test substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes considered as treatment-related were limited to both sexes at 300 mg/kg bw/day. After the end of the treatment period, centrilobular slight hepatocyte hypertrophy was observed in 2/5 males and 3/5 females in the 300 mg/kg bw/day group. This change may be related to the increased relative liver weight in the male 300 mg/kg bw/day group. The effect is considered to be treatment-related, but adaptive as no changes in the liver was observed in the recovery animals. In the same group, a slight fold of the duodenal mucosa was observed in one male. The relevance of this observation in unclear.
In the 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. The erosion effects seen in the GI tract in several animals was related to the corrosive effect of the substance.
See Table 8 under “Any other information on results including tables” and Table 4 of the attached study summary.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical signs
gross pathology
mortality
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
central nervous system
Organ:
other: CNS
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
yes

Table 1: Body weight changes in male rats after recovery period treated orally with PHF in the combined repeated dose and reproductive/developmental toxicity screening test.

Sex

Dose level (mg/kg)

Recovery period

0

300

Male

Number of animals

5

5

Day 50

543.8 ± 29.1

497.4 ± 26.6*

Day 56

552.4 ± 30.7

506.8 ± 27.4*

Values are expressed as Mean ± S.D.

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

 

Table 2: Body weight changes in female rats during lactation period treated orally with PHF in the combined repeated dose and reproductive/developmental toxicity screening test.

Sex

Dose level (mg/kg)

Administration period

0

10

60

300

Female

Number of animals

5

5

5

5

Day 4 lactation

335.8±25.0

341.1 ± 33.4

356.0 ± 25.4

295.7 ± 11.3

Values are expressed as Mean ± S.D.

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

 

Table 3: Food consumption changes in male rats after recovery period treated orally with PHF in the combined repeated dose and reproductive/developmental toxicity screening test.

Sex

Dose level (mg/kg)

Recovery period

0

300

Male

Number of animals

5

5

Day 50

31.16 ± 1.66

25.60 ± 2.45**

Day 54

30.42 ± 1.15

25.72 ± 2.09**

Values are expressed as Mean ± S.D.

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

 

 

Table 4: Food consumption changes in female 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

Female

Number of animals

12

12

12

12

5

5

Day 14

19.9 ± 1.5

20.8 ± 2.4

22.6 ± 1.8**

20.9 ± 1.7

 

 

Number of animals

12

12

11

10

 

 

Gestation Day 20

26.4 ± 1.7

27.2 ± 3.1

27.5 ± 3.6

21.4 ± 3.0**

 

 

Number of animals

12

12

11

6

 

 

Lactation Day 4

36.2 ± 6.1

36.7 ± 4.4

37.8 ± 4.9

23.3 ± 6.7**

 

 

Number of animals

 

 

 

 

5

5

Day 56

 

 

 

 

22.24 ± 4.91

15.80 ± 1.22*

Values are expressed as Mean ± S.D.

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

 

 

Table 5: 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

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

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

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

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)

35.36 ± 0.30

35.38 ± 0.42

35.56 ± 0.67

35.14 ± 0.42

35.58 ± 0.48

35.44 ± 0.32

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

Female

Number of animals

5

5

5

5

5

5

Reticulocyte (%)

8.622 ± 1.222

8.624 ± 3.069

6.466 ± 1.999

9.446 ± 4.226

2.910 ± 0.984

2.308 ± 0.895

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

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*

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**

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

Values are expressed as Mean ± S.D.

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

Table 7: 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*

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*

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

Relative organ weight 

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

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*

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

Relative organ weight 

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**

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

 

Table 8: Histological findings in rats treated orally with PHF in the combined repeated dose and reproductive/developmental toxicity screening test

 

Sex

Male

Female

Organ

 Findings

Dose level (mg/kg)

After administration

After recovery period

After administration

After recovery period

0

10

60

300

0

300

0

10

60

300

0

300

 

Numer of animals

5

5

5

5

5

5

5

5

5

5

5

5

Liver

 Hypertrophy, hepatocyte, centrilobular

Grade

1

0

0

0

2

0

0

0

0

0

3

0

0

Small intestine, duodenum

 Erosion, mucosa

Grade

1

0

0

0

1

0

0

0

0

0

0

0

0

Executive summary:

In accordance with OECD guideline 422 (Combined repeated dose and reproductive/developmental toxicity screening test), the test item was studied for oral toxicity in rats at doses of 10, 60, and 300 mg/kg bw/day. The duration of administration was 42 days for males and 42-52 days for females. An additional satellite group for the control and high-dose group was included, with a post-exposure recovery period of 14 days.

Treatment-related changes were observed at 300 mg/kg bw/day. At this dose, clinical signs attributed to central nervous system (CNS) depression and anesthetic action of the test substance were observed in both sexes shortly after dosing (0 -30 minutes). The adverse effects had disappeared by 3 hours after dosing. 1/12 females administered 300 mg/kg bw/day died during delivery following dosing on day 24 of gestation. Prior to death, signs of CNS depression were also observed in the animal that died during the treatment period.

Detailed clinical observations also revealed significant signs of CNS depression (such as prone position, abnormal gait and crawling) for both males and females at the high dose. Onset of these signs in females was already noted during the first week of dosing, but for males did not start before week 2 or 3. As for the functional observation battery, high dose animals showed abnormal reactivity to stimuli, reduced grip strength and reduced motor activity. All of these changes recovered after the recovery period and were therefore considered reversible changes. However, these were considered toxicologically relevant adverse effects.

In females at 300 mg/kg bw/day, body weight gain and food consumption decreased significantly (lactation day 4) or non-significantly (by approximately 10%) in the late part of gestation until day 4 of lactation, compared with the control group. Body weight gain of males in this group also decreased (by approximately 10%, non-significant) late in the administration period, compared with the control group. Body weight gain was significantly reduced, compared with the control group, during the recovery period. In females administered 300 mg/kg bw/day food consumption was significantly reduced on gestation day 20 and lactation day 4, compared with the control females. The recovery group also showed a significant reduction in food consumption at the end of the recovery period, compared with the control group. In males administered 300 mg/kg bw/day food consumption was comparable to that of the control group during the administration period, but it was significantly decreased during the recovery period.

Increased triglyceride was found in the male groups at 60 mg/kg bw/day or higher and one female 300 mg/kg bw/day. Although relation of this change to treatment was not clear, it was reversible and not accompanied by any histopathological changes, therefore it was considered not toxicologically relevant.

At 300 mg/kg bw/day, hypertrophy of centrilobular hepatocytes was observed in both sexes and liver weight was significantly increased in males. There were no fatty degeneration or any increases in liver enzymes indicating liver damage, and it was a reversible change, as no similar effects were seen in the liver of the high-dose satellite animals. Therefore, this change was considered to be an adaptive change rather than adverse. At 300 mg/kg bw/day, one male sacrificed at the planned termination point showed erosion of the duodenal mucosa, and the female that died during the administration period had erosion with hemorrhage in the fundic mucosa and erosion of the duodenal mucosa with hypertrophy of the duodenal glands. The substance is corrosive (see section 7.3.1). Therefore, erosion in the stomach and duodenal mucosa was considered to be caused by the corrosive/irritating property of the test item.

 

The NOAEL for repeated dose toxicity of the test item was considered to be 60 mg/kg bw/day for males and females based on a death in females, clinical signs attributed to CNS depression and anesthetic action, decreased body weight gain and irritative changes in the gastrointestinal tract (mainly in duodenal mucosa) observed in both sexes at 300 mg/kg bw/day.

Accordingly, the test item was classified for specific target organ toxicity following repeated exposure, STOT RE 2, H373, according to Regulation (EC) No. 1272/2008 (CLP).

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
March 22, 1996
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
yes
Remarks:
Pups were terminated on postnatal day 4, the T3, T4, TSH levels were not measured, anogenital distance was ot measured, nipple retention was not recorded.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1,1,1,3,3,3-hexafluoropropan-2-ol
EC Number:
213-059-4
EC Name:
1,1,1,3,3,3-hexafluoropropan-2-ol
Cas Number:
920-66-1
Molecular formula:
C3H2F6O
IUPAC Name:
1,1,1,3,3,3-hexafluoropropan-2-ol
Test material form:
liquid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: sealed in bottle in a fridge
- Stability under test conditions: verified by IR at the beginning and at the end of the study

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc., Yokohama, Japan
- Age at study initiation: 9 weeks old
- Weight at study initiation: 317 - 361 g (male), 199 - 251 g (female)
- Fasting period before study: none
- Housing: 1 male and 1 female was housed in one stainless-steel wire-meshed cages except for gestation and lactation periods. During gestation and lactation periods, one female was housed in a polycarbonate cage.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: one week including quarantine period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.8 - 23.6
- Humidity (%): 49.8 - 61.8
- Air changes (per hr): 6 - 20
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From 12 Sep 2007 To 13 Nov 2007

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dosing solutions were prepared daily before administration. Using purified water as a vehicle, a 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/mL.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Up to 14 days.
- Proof of pregnancy: Vaginal plug referred to as day 0 of pregnancy.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Because of the 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 postpartum until lactation day 4.
Frequency of treatment:
7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
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/Satellite groups:
Control and high dose groups: 5 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a 14-day preliminary study, a dose of 500 mg/kg bw/day was considered too high for the main study because of poor general conditions for the test animals. As a consequence, the top dose in the main study was set to 300 mg/kg bw/day. The mid- and low doses were set as 60 and 10 mg/kg bw/day, respectively.
- Rationale for selecting satellite groups: Satellite groups for recovery study were set for control and high dose groups to assess the recovery from observed effects.
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Parental animals: Observations and examinations:
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: the first observation after the first administration, thereafter weekly. Detailed clinical observations were recorded after administration, however, no information how long after dosing is available.

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

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

FOOD EFFICIENCY: No
- 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 h
- How many animals: 5 per sex per group
- Parameters checked: RBC, hemoglobin, hematocrit, reticulocyte, mean cell volume (MCV), mean cell hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet, prothrombin time (PT), activated partial thromboplastin time (APTT), white blood cell (WBC), lymphocyte, neutro phil, eosinophil, basophil, and monocyte.

CLINICAL CHEMISTRY: 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 h
- How many animals: 5 per sex per group
- Parameters checked: aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), gamma-glutamyl transferase (GT), alkaline phosphatase (ALP), toal bilirubin, urea nitrogen, creatinine, glucose, total cholesterol, triglyceride, total protein, albumin, albumin to globulin (A/G) ratio, calcium, inorganic phosphate, sodium, potassium, and chloride.

URINALYSIS: Yes, 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 and urobilinogen.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: the day before start of administration, once a week during administration
- Dose groups that were examined: all groups
- Battery of functions tested: sensory activity, grip strength, motor activity, and reactivity to stimuli.
Oestrous cyclicity (parental animals):
Estrous cyclicity observation: performed daily from the first day of administration until confirmation of copulation.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testis weight and epididymis weight.
Litter observations:
The following parameters were examined in F1 offspring:
Number of pups delivered, Number of live pups on day 0, sex ratio, Number of live pups on day 4, body weight on day 0 and body weight on day 4.
Postmortem examinations (parental animals):
SACRIFICE:
Male animals: Rats were euthanized by exsanguination under anesthesia on the day after the last administration.
Maternal animals: Rats were euthanized by exsanguination under anesthesia on Day 4 of lactation.

GROSS NECROPSY: Yes, Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

GROSS PATHOLOGY: Yes; spleen, liver, heart, lymph node, mandibular, lymph node, mesenteric, thymus, bone marrow, femur, trachea, lung (and bronchus), stomach, small intestine, duodenum, small intestine, jejunum, small intestine, ileum, large intestine, cecum, colon, rectum, kidney, urinary bladder, ovary, uterus, vagina, mammary gland, pituitary, thyroid, parathyroid, adrenal, brain, spinal cord, and sciatic nerve.

ORGAN WEIGHTS: Yes; brain, heart, liver, kidney, adrenal gland, thymus, spleen, testis and epididymis.

HISTOPATHOLOGY: Yes
Control and 300 mg/kg bw/day groups: brain, pituitary gland, thymus, lymph node, trachea, lung, stomach, intestine (duodenum, jejunum, ileum, cecum, colon, rectum), thyroid gland, parathyroid (bilateral), heart, liver , spleen, kidney (bilateral), adrenal (bilateral), bladder, testis (bilateral), epididymis (bilateral), seminal vesicle (including coagulation gland), prostate abdominal lobe, ovary (bilateral), uterus, sputum, bone marrow of right femur, right sciatic nerve, spinal cord, and gross abnormal area.
All groups: liver, small intestine(duodenum), testes, and epididymides.
The above listed organs and tissues of all animals were collected, fixed with 10 vol% neutral phosphate buffered formalin, and stored. Male testis and epididymis were fixed with bouin solution and stored in 10 vol% neutral phosphate buffered formalin solution.
Postmortem examinations (offspring):
SACRIFICE: The F1 offspring were sacrificed on lactation Day 4.
GROSS NECROPSY: On lactation Day 4, the pups were euthanized by exsanguination under anesthesia and gross internal examinations were performed.
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.
Reproductive indices:
Mating index (%) = (Number of pairs with successful mating/number of pairs examined)×100
Fertility index (%) = (Number of pregnant animals/number of pairs with successful mating)×100
Gestation index (%) = (Number of females with live pups/number of pregnant females)×100
Implantation index (%) = (Number of implantation sites/number of corpora lutea)×100
Delivery index (%) = (Number of pups delivered/number of implantation sites)×100
Offspring viability indices:
Live birth index (%) = (Number of live pups on day 0/number of pups delivered)×100
Viability index (%) = (Number of live pups on day 4/number of live pups on day 0)×100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No abnormality was found in the 10 and 60 mg/kg bw/day groups of both sexes. In the 300 mg/kg bw/day group, prone position and a decrease in locomotor activity were observed in both sexes. These changes were seen throughout the administration period in 1/12 to 9/12 animals from immediately after dosing and disappeared by 3 to 6 hours after dosing. No clinical signs were noted during the recovery period. The effects were considered to be treatment-related and toxiclogically relevant.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
One female in the 300 mg/kg bw/day group 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 changes:
effects observed, treatment-related
Description (incidence and severity):
There were no changes in body weight and body weight gain in male and female groups of 10 and 60 mg/kg bw/day compared with control. In females at 300 mg/kg bw/day, body weight gain showed 10% decrease from the late gestation until day 4 of lactation compared with control (see Table 2 under “Any other information on results including tables”). This change was statistically significant on lactation day 4. Body weight gains of males in the 300 mg/kg bw/day group showed almost 9% decrease compared with control after Day 22 (see Table 1 under “Any other information on results including tables”). This change was significant on Day 50 and 56. Although the body weight remained significant low during the recovery period, the weight gain during the last week of the recovery males was similar to that in the control group. The females in the recovery group did not gain weight from Day 42 to 56. The effects on body weight are treatment-related and toxicologically relevant, adverse effect.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There were no changes in food consumption in male and female groups of 10 and 60 mg/kg bw/day compared with control. Food consumption in males at 300 mg/kg bw/day was similar to that of the control group during the administration period. The food consumption was significantly reduced (by 20%) during the recovery period on Day 50 and Day 54 in males at 300 mg/kg bw/day group compared with the control. See Table 3 under “Any other information on results including tables”. The food consumption in the females showed a 20% decrease on Gestation Day 20 and 35% decrease on Lactation Day 4 at 300 mg/kg bw/day group compared with the control. See Table 4 under “Any other information on results including tables”. In the recovery group, the reduced food consumption was significant in females in the 300 mg/kg bw/day group on Day 56, during the recovery period. The effects are treatment-related and toxicologically relevant, adverse effect.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No change was noted at 10 and 60 mg/kg bw/day in both sexes compared with control. At 300 mg/kg bw/day, 35% decreased reticulocyte count and 25% decreased platelet count was noted in males at the end of the administration compared with control. The effects were not considered to be toxicologically relevant, since there were no histological changes and the changes were only observed in the males. A 40% decreased white blood cell count in females at 300 mg/kg bw/day was noted at the end of the administration compared with control. The effects were not considered to be toxicologically relevant, since there were no histological changes and the changes were only observed in the females. At the end of the recovery period, 8% increased red blood cell and 7% decresased MCV and 7% decresased MCH was noted in females of the 300 mg/kg bw/day group compared with control. See Table 5 under “Any other information on results including tables”. These changes were also not considered to be toxicologically relevant since the changes were not observed at the end of the administration period.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A 450% increased triglyceride level was noted in the male 300 mg/kg bw/day group compared with control. Although statistically not significant, 180% and 160% increase in triglyceride was observed in the male 60 mg/kg bw/day and 300 mg/kg bw/day group compared with control, respectively. For the triglycerides, this may be related to the test substance causing increased metabolic activity in the liver, also seen as an increase in liver weight in the high-dose males, and therefore treatment-related. However it is reversible and therefore considered to be an adaptive response. Urea nitrogen showed 18% increase in males at 300 mg/kg bw/day compared with control; however, creatinine did not differ from the control value without any effects on the renal function in other examinations. This change was not considered as treatment-related effect. In female group at 300 mg/kg bw/day, decreases in calcium (8% decrease) and potassium (12% decrease) were observed in comparison with control. The potassium values was within ± 2 S.D of the historical control range. This change was considered as an incidental change. With regard to calcium, the change was minimal and was not observed at the end of the recovery period. Therefore, the changes in calcium level is considered to be incidental. The ASAT values showed 15 – 20% decrease in females in all administration groups compared with control. When compared with the historical control range of the test facility, two animals of the control group had higher values (mean + 2 S.D.). Furthermore the resultswere non-significant, and this change was not considered a treatment-related effect. Males in the 60 mg/kg bw/day group showed 40% increased ASAT value compared with control. This change was not considered as the treatmentrelated effect since there was no dose-depending manner noted. Females in the 60 mg/kg bw/day group showed increased total cholesterol compared with control. However, this change was not considered as the treatment-related effect since a dose-depended increase was not observed. At the end of recovery period, the satellite females in the 300 mg/kg bw/day group showed non-significant changes in ASAT (30% decrease), and ALAT (200% decrease) compared with control. However, the same change was not observed at the end of administration period, and as a result of comparison with the background data of the test facility, all cases were within the range of the mean ± 2 S.D. of the background data. It was considered as an incidental change. See table 6 under “Any other information on results including tables”.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
No change was noted up to 60 mg/kg bw/day in both sexes compared with control. In the 300 mg/kg bw/day groups, prone position (1 – 6 males in 12 males between week 3 and 6, 1 – 3 females in 17 females between week 1 and 3, 5 and 6), lateral position (2/12 males at week 3, 1/17 female at week 5), crawling position (1 – 3/12 males at week 2 and 4, 1 – 3/17 females at week 1, 2, 3, 5 and 6) andabnormal gait in both sexes (1 – 10/12 males during week 2 and 6, 1 – 6/17 females during week 1 and 6) were recorded. As for the functional test, males in the 300 mg/kg bw/day group (within 5 males) showed abnormal reactivity to stimuli (2 male without reaction to approach response and touch response, 1 male without reaction to auditory reponse, 1 male with slight bad aerial righting reaction, 2 males with disapperance of aerial righting reaction). In the 300 mg/kg bw/day group, grip strength was decreased in males (38% for forelimb) and females (36% for forelimb and 36% for hindlimb). Motor activity after the administration between 10 and 50 minutes was significantly decreased (90% decrease in males and 98% decrease in female). These effects on behaviour and functional parameters were not observed during the recovery period, indicating they were reversible changes over time. Aseffects were serious, and consistently observed in mutiple parameters in both sexes from 2-3 weeks after administration started, In the high-dose group, these are considered to be treatment-related, toxicologically relevant adverse effects.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes considered as treatment-related were limited to both sexes at 300 mg/kg bw/day. After the end of the treatment period, centrilobular slight hepatocyte hypertrophy was observed in 2/5 males and 3/5 females in the 300 mg/kg bw/day group. This change may be related to the increased relative liver weight in the male 300 mg/kg bw/day group. The effect is considered to be treatment-related, but adaptive as no changes in the liver was observed in the recovery animals. In the same group, a slight fold of the duodenal mucosa was observed in one male. The relevance of this observation in unclear. In the 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. The erosion effects seen in the GI tract in several animals was related to the corrosive effect of the substance. See Table 8 under “Any other information on results including tables” and Table 4 of the attached study summary.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
The mean length of the oestrous cycle was significantly increased in the high-dose females. The oestrous cycle was 4.25 ± 0.40, 4.09 ± 0.30, 4.22 ± 0.44 and 5.23 ± 0.68 in the control, 10, 60 and 300 mg/kg bw/day groups, respectively. In the 300 mg/kg bw/day group 4/12 females had an oestrous cycle length of 6 days, contributing to the increased mean value. This observation is considered to be treatment-related and toxicologically relevant. See the Table 1 under the “Any other information on results including tables”.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
The mean length of the gestation was significantly increased in the high-dose females. The mean gestation length was 22.3 ± 0.5, 22.6 ± 0.5, 22.7 ± 0.5 and 23.4 ± 0.7 in the control, 10, 60 and 300 mg/kg bw/day groups, respectively. Effects were also seen in the delivery index (17% decrease), although it was not significant and with a large standard deviation. These changes were considered as treatment-related and toxicologically relevant. See Table 2 under the “Any other information on results including tables”.
The parameters on fertility index, pairing days until mating, number of corpora lutea, number of implantation sites, number of pups delivered did not show any treatment-related changes.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
LOAEL
Remarks:
fertility
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed on fertility and reproductive performance
Key result
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
gross pathology
other: CNS
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
central nervous system
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
yes

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The loss of suckling action was observed in pups in the 300 mg/kg bw/day group. This was regarded as a treatment-related and toxicologically relevant adverse effect. See Table 3 under “Any other information on results including tables”.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The mean number of live pups on day 0 was decreased in the 300 mg/kg bw/day group, with a significantly reduced vialbility index compared with the control. The mean delivery index showed a 30% decrease in the 300 mg/kg bw/day group, compared with control. The mean number of live pups on day 4 was 14.3 ± 2.1, 14.9 ± 1.6, 14.2 ± 3.4 and 5.2 ± 5.3 in the control, 10, 60 and 300 mg/kg bw/day groups, respectively. The mean viability index on day 4 was also significantly decreased (60%) in the 300 mg/kg bw/day group, compared with control. These changes were considered as a treatment-related and toxicologically relevant effect on development. See Table 6 of the attached study summary under "Attached background material”.

Futhermore, 73 pups in the 300 mg/kg bw/day group died during the lactation period, whereas 3, 3, and 4 pups were dead in the control, 10, and 60 mg/kg bw/day group, respectively, until lactation Day 4.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
On lactation Day 4, the mean body weight of pups was significantly decreased (32% for male and 25% for female) compared with control.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
20 and 22 pups or fetuses had generalized edema and protruding tongue, respectively. These effects were observed only in the pups of the dam that died on Day 24 and in the pups of dams in the 300 mg/kg bw/day group that died between postnatal day 0 and 4. During the lactation period, 2 and 5 dead pups had generalized edema and protruding tongue on lactation Day 0. These effects were not observed in the later lactation period. The generalised edema is probably a systemic effect in the pups, caused by the treatment of the dam. Protruding tongue is regarded as a treatment-related, but it is not clear what causes it.
1/26 male pup had black contents in the intestine and 4 pups (3/26 males and 1/21 female) had dark red contents in the stomach and intestine; all of dams in the 300 mg/kg bw/day group. Since these changes were not observed in the other groups, it is considered to be treatment-related.
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

All pups of three dams (No. 50405, 50409, 50410) in the 300 mg/kg bw/day group died between postnatal day 0 and 4.

No. 50405:
Decreased locomotor activity and prone position were observed after administration on lactation Day 0, and all pups died in the afternoon of the day.

No. 50409:
The administration was performed during the delivery on gestation Day 24. However, the dam had not yet delivered even in the evening. In addition, after the administration on gestation Day 24, prone position and decreased locomotor activity were observed. The delivered pups were not cared for by the dam. Three hours after administration, the symptoms in the dam had resolved, but two pups died. On the following day, delivery was completed, and there were 2 surviving pups: 1 died on lactation Day 1 and 1 died on lactation Day 2.

No. 50410:
After the administration on gestation Day 23 (during delivery), prone position and decreased locomotor activity were observed. The delivered pups were not cared for. Three hours after the administration, the symptoms in the dam had resolved, but 6 pups died. Thereafter, the dam showed decreased locomotor activity and prone position after administration, and did not nurse the litter on lactation Day 2. All pups died by lactation Day 4.

One dam (No. 50402) in the 300 mg/kg bw/day group died during the delivery.

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
LOAEL
Remarks:
developmental
Generation:
F1
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

Table 1 Fertility and pregnancy data in rats treated orally with 2-Propanol, 1,1,1,3,3,3-hexafluoro- in the combined repeated dose and reproductive/developmental toxicity screening test

Dose level (mg/kg)

Administration period

0

10

60

300

Number of pairs examined

12

12

12

12

Estrous cycle duration

 4.25 ± 0.40 

4.09 ± 0.30

4.22 ± 0.44

5.23 ± 0.68**

Values are expressed as Mean ± S.D.

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

Table 2 Delivery and litter data in rats treated orally with 2-Propanol, 1,1,1,3,3,3-hexafluoro- the combined repeated dose and reproductive/developmental toxicity screening test

Dose level (mg/kg)

Administration period

0

10

60

300

Number of females examined

12

12

11

10

Gestation length (days)  

22.3 ± 0.5 

22.6 ± 0.5 

22.7 ± 0.5 

23.4 ± 0.7**

Number of live pups on day 4

14.3 ± 2.1 

14.9 ± 1.6 

14.2 ± 3.4 

5.2 ± 5.3**

Live birth index (%) a)

100.00 ± 0.00 

99.44 ± 1.93 

98.10 ± 3.30 

70.20 ± 33.60**

Viability index on day 4 (%) b)

98.23 ± 3.28 

99.07 ± 2.18 

99.44 ± 1.87 

39.71 ± 40.52**

Body weight of pups (g)

on day 4 male

10.4 ± 0.9 

10.7 ± 1.0 

11.1 ± 2.2 

7.1 ± 2.3*

on day 4 female

9.9 ± 1.0 

10.2 ± 1.0 

10.5 ± 2.2 

7.4 ± 1.5*

a) Live birth index (%) = (Number of live pups on day 0/number of pups delivered)×100

b) Viability index (%) = (Number of live pups on day 4/number of live pups on day 0)×100

Values are expressed as Mean ±S.D.

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

Table 3 Necropsy findings in F1 offspring

Dose (mg/kg)

 

Scheduled Sacrifice

Dead

Day 4

0

1

2

3

4

M

F

M

F

U

M

F

M

F

M

F

M

F

0

Total*

91

80

 

 

 

 

 

 

1

 

 

1

1

 

No abnormality

91

80

 

 

 

 

 

 

0

 

 

0

0

 

Generized edema

0

0

 

 

 

 

 

 

0

 

 

0

0

 

Portruding tongue

0

0

 

 

 

 

 

 

0

 

 

0

0

 

Los of suckling

0

0

 

 

 

 

 

 

0

 

 

0

1

10

Total*

91

88

 

1

 

1

 

 

 

 

 

 

1

 

No abnormality

91

88

 

0

 

0

 

 

 

 

 

 

0

 

Generized edema

0

0

 

0

 

0

 

 

 

 

 

 

0

 

Portruding tongue

0

0

 

0

 

0

 

 

 

 

 

 

0

 

Loss of suckling

0

0

 

0

 

0

 

 

 

 

 

 

 

60

Total*

70

86

2

1

 

 

 

 

 

 

 

1

 

 

No abnormality

70

86

0

0

 

 

 

 

 

 

 

0

 

 

Generized edema

0

0

0

0

 

 

 

 

 

 

 

0

 

 

Portruding tongue

0

0

0

0

 

 

 

 

 

 

 

0

 

 

Loss of suckling

0

0

1

1

 

 

 

 

 

 

 

1

 

300

Total*

26

21

10

16

3

15

13

3

4

5

1

3

 

 

No abnormality

21

20

0

0

0

1

3

0

1

0

0

0

 

 

Genralized edema

0

0

2

0

0

0

0

0

0

0

0

0

 

 

Portruding tongue

0

0

4

1

0

0

0

0

0

0

0

0

 

 

Abnormal contents, black, intestine

1

0

1

0

0

0

0

0

0

0

0

0

 

 

Abnormal contends, dark red, stomack and intestine

3

1

0

0

0

0

0

0

0

0

0

0

 

 

Loss of suckling

0

0

5

11

0

4

2

0

0

2

0

0

 

*: number of offsprings examined

M: male, F: female, U: unknown

Applicant's summary and conclusion

Executive summary:

In accordance with OECD guideline 422 (Combined repeated dose and reproductive/developmental toxicity screening test), the test item was studied for its reproductive toxicity potential following oral dosing at doses of 10, 60, and 300 mg/kg bw/day. The duration of administration was 42 days for males and 42-52 days for females. An additional satellite group for the control and high-dose group was included, with a post-exposure recovery period of 14 days.

Signs of toxicity were restricted to the 300 mg/kg bw/day group. The estrous cycle length was prolonged to 6 days in 4 dams and the mean estrous cycle length in this group was significantly prolonged (5.23 ± 0.68 days), compared with the control (4.25 ± 0.40 days). The copulation index was 83.3% (10/12 animals) at 300 mg/kg bw/day while for all other groups it was 100%. During the gestation period, none of the high-dose dams completed the delivery on day 22 of gestation and one of them showed dystocia as it delivered on day 25 of gestation and finally died during delivery. Accordingly, the mean gestation length was significantly prolonged in this group, compared with control. The live birth index was significantly decreased (70% compared to 100% in the control) and postnatal pup survival was decreased; resulting in reduced numbers of pups (5.2 compared to 14.2 pups/litter in the control) on day 4 post partum. Pup body weights at 300 mg/kg bw/day were similar to those of the control at first litter check on Day 0 postpartum but were significantly reduced on Day 4 postpartum. 3/10 dams lost the entire litter between Day 0 postpartum and Day 4 postpartum. These dams showed poor maternal care towards the pups.

For the foetuses of the dam that died during delivery and the pups that died before Day 4 postpartum, in the high dose group, generalized edema and protruding tongue were observed in 20 fetuses/pups and in 22 fetuses/pups, respectively. In this group, increased pleural fluid was observed in 3 pups. Blackish and/or dark reddish abnormal contents in the intestine were observed in 6 pups. These effects were considered to be treatment-related and toxicologically relevant adverse effects.

Based on these data the NOAEL for parental general toxicity and reproductive toxicity was considered to be 60 mg/kg bw/day. The NOAEL for developmental toxicity was considered to be 60 mg/kg bw/day.

 

The noted effects on reproductive parameters in dams and in the F1 offspring at 300 mg/kg bw/day were considered not entirely attributable to the noted maternal toxicity. Therefore the test item was classified as toxic to reproduction, Rep. 2, H361, according to Regulation (EC) No. 1272/2008 (CLP).

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