Tetradecafluorohexane

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

April 7 — September 3, 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

other company data

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Principles of method if other than guideline:

AIM: To assess effects of AF0150 IV injection on the pregnant/lactating female and on development of the conceptus and the offspring (through sexual maturity)
Methods:
AF0150 solution and saline (0.9% sodium chloride for injection, as a negative control) were administered daily by IV injection via a lateral caudal vein (dilating with warm water, if necessary) from gestation day 6 through lactation day 20. The AF0150 dosages were 50, 100 and 200 mg/kg/day for assigned groups, and 5 ml/kg/day of saline for control group.
Maternal (Fo) Observations
Clinical Signs: All animals were observed twice a day for mortality and moribundity, and detailed clinical signs were recorded daily till scheduled sacrifice day (Lactation day 21).
Food consumption: Individual maternal food consumption was recorded on gestation days 0, 6, 9, 12, 15, 18 and 20, and on lactation days 1, 4, 7, 14 and 21; reported as g/animal/day and g/kg/day for each corresponding body weight change.
Body Weights: Maternal body weights were recorded on gestation days 0, 6, 9, 12, 15, 18 and 20, and on lactation day 1, 4, 7 14 and 21. Group mean body weight was calculated for each time point. Mean body weight changes were calculated for each corresponding interval and also for gestation days 0-20 and for lactation days 1-21.
Pregnancy Duration and Parturition: all surviving animals were allowed to delivery naturally and rear their offspring to weaning. The pregnancy duration was defined as days from gestation day 0 to initiation of parturition. The day of complete delivery was designated PND 0 [PND was not defined, maybe Post-Natal Day]. The number, sex and malformation of stillborn and live pups in each litter were recorded at the end of parturition.
Necropsy: all animals, including those that were scheduled for termination (on lactation day 21), unscheduled death, and those failed to delivery or had total litter loss, were subjected to necropsy. The thoracic, abdominal and pelvic cavities and contents were examined. The numbers of former implantation sites or corpora lutea (for unscheduled death) were recorded.

Offspring (FI) Observations
Clinical Signs: each litter was examined daily for appearance, behavior, survival and death. Each pup received a detail physical examination on PND 1, 4, 4, 14 and 21 and weekly thereafter till necropsy. Pup sexes were determined on PND 0, 4 and 21. Eight pups per litter (4 each sex when possible) were randomly selected, weighed and sacrificed on PND 4. Liner parameters were processed

Body Weights: individual body weight was recorded on PND 1, 4, 7, 14 and 21, and weekly thereafter tilt necropsy.
Offspring Development: Each dam and litter remained together till weaning on PND 21. Fifty pups (25 each sex) between 6-10 days old were randomly selected from each of 4 groups (saline, 50, 100, and 200 mg/kg/day AF0150) for assessment of developmental landmarks and reproductive performance. The remaining pups were sacrificed and necropsied on PND 21. The following parameters were observed in the selected pups:
1. Balanopreputial Separation: male pups were observed daily for balanopreputial separation beginning on PND 40.
2. Vaginal Perforation: female pups were observed daily for vaginal perforation beginning on PND 30.
3. Auditory Startle Test: 10 pups/sex/group on PND 21 and 60 (±5 days) received an auditory response test in the sound chamber using an automatic Auditory Startle Response System. The responses or movements of animals to the tested noise bursts were monitored by weight¬sensitive platforms on the bottom surface of the sound chamber. Mean peak amplitude (grams), latency to peak (mSec), response duration (mSec) and average response (grams) on each block (1-5) of 10 trials were recorded.
4. Motor Activity: 10 pups/sex/group on PND 13, 17, 21 and 60 (±2 days) were subjected to
motor activity test using the grem Animal Activity System. The animals were
placed in a clear plastic rectangular cage surrounded with series of infrared photobeams. Both fine (interrupting one or two adjacent photobeams) and ambulatory (interrupting 3 or more consecutive photobeams) motor activities were collected.
5. 01Maze Swimming Trials: learning and memory ability of pups (10 pups/sex/group) were
evaivated using the maze swimming test in a water-filled, 8-unit T-maze system. The first test was conducted between PND 20 and 24, and the second test was between PND 60 and 64. The mean number of errors and the mean escape time for each trail were considered measures of maze acquisition.
6. Reproductive Performance and Fertility of Offspring: the Fi animals at 87-91 days old were paired on a 1:1 basis within each treatment group but avoiding sibling pairing. Positive evidence of mating was confirmed by the presence of a copulatory plug or sperm in a vaginal smear, and gestation day 0 was defined as the day that positive mating was identified. Clinical signs, estrous cycles, body weights and gravid uterine weights were observed. On gestation day 20, all maternal animals were subjected to complete necropsy and fetuses (F2) were examined for malformation and variations. Female/male mating index and fertility index, post-implantation loss/liter were calculated, as described in the fertility study procedure.

GLP compliance:

yes

Remarks:

Attested by US-FDA in the context pf NDA 21-191

Test material

Test material information

Constituent 1

Reference substance name:

Tetradecafluorohexane

EC Number:

206-585-0

EC Name:

Tetradecafluorohexane

Cas Number:

355-42-0

Molecular formula:

C6F14

IUPAC Name:

tetradecafluorohexane

Test material form:

liquid

Remarks:

Preparation for iv injection

Details on test material:

Imagent® Kit for the preparation of perflexane lipid microspheres for injectable suspension,
is a sterile, non-pyrogenic white powder with a diluted perflexane headspace that, after
reconstitution into a suspension of microspheres, is used for contrast enhancement during the
indicated ultrasound imaging procedures.
The contents of the 200 mg Imagent powder vial are sterile and non-pyrogenic. Each vial
of Imagent® powder contains 9.2 mg 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC);
75 mg hydroxyethyl starch; 2.1 mg poloxamer 188; 75 mg sodium chloride; and 36 mg
sodium phosphate buffer in a vial filled with a mixture of 17% v/v perflexane vapor in
nitrogen.
After reconstitution with 10 mL of the provided Sterile Water for Injection, USP, the
contents of the vial yield an opaque white suspension for injection. The reconstituted
suspension must be withdrawn from the vial with the supplied vented 5 µm filter dispensing
pin.
Each mL of reconstituted aqueous suspension contains a maximum of 13.7 x 108
microspheres, 92 µg perflexane, 0.92 mg DMPC; 7.5 mg hydroxyethyl starch; and 0.21 mg
poloxamer 188. The reconstituted product is iso-osmolar and has a pH between 6.7 to 7.7.
Table 1. Microsphere Size Distribution
DIAMETER
Mean Volume Weighted Median: 6 µm
Number per mL
Mean (% of Total)
ALL SIZES (Total) 5.9-13.7 x 108
(100%)
<3 µm 7 x 108
(78.8%)
3 - 10 µm 2 x 108
(21.0 %)
>10 µm 0.01 x 108
(0.2 %)
Upper limit 20 µm
The active moiety, the microsphere, comprises two critical components: perflexane, the
gaseous component, and DMPC, the lipid membrane component.
Perflexane is chemically characterized as n-perfluorohexane with a molecular weight of 338
atomic mass units and an empirical formula of C6F14. Perflexane has the following structural
formula:
FF FF F
F
F
F F F
F
F
F F
DMPC is a semi-synthetic (not of animal origin) phospholipid and is chemically
characterized as 1, 2,-dimyristoyl-sn-glycero-3-phosphocholine with a molecular weight of
678 atomic mass units and an empirical formula of C36H72NO8P. DMPC has the following
structural formula:
O H C
O
O CH2
H2C
O
O
P
O O
O
N(CH3)3 -
+
Imagent Kit for the Preparation of Perflexane-Lipid Microspheres Injectable Suspension is
supplied for single-use and each kit contains a 10-mL glass vial containing 200 mg of
Imagent powder, a 20-mL plastic vial of Sterile Water for Injection, a 10-mL disposable
plastic sterile syringe, a sterile, vented 5 µm filter dispensing pin, and a package insert.
The powder vial must be reconstituted with 10 mL supplied Sterile Water for Injection and
then withdrawn from the vial with the provided vented 5 µm filter dispensing pin as
described under DOSAGE AND ADMINISTRATION – Drug Handling and Preparation.

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)BR

Details on species / strain selection:

Crl:CD(SD)BR rats (100 each sex, 45 days old)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Crl:CD(SD)BR female rats (125, sexually mature and virgin, 70 days old)
Standard procedures were
followed for housing, handling, feeding and care of the animals. Alter acclimation for 11 days, animals (about 12 weeks old) meeting good health and acceptable body weight requirements (minimum 220 g) were paired with resident male rats (the same strain, source and sexually mature) [the ratio was not specified] for breeding. The gestation day 0 was defined as the day that positive evidence of mating was identified by the presence of a copulatory plug or sperm in a vaginal smear. The female rats on gestation day 0 were randomly assigned to 4 groups, 25/group (body weight of 214-270 g). All animals were individually housed through the study, except during mating with the resident male rats.

Administration / exposure

Route of administration:

intravenous

Vehicle:

water

Details on exposure:

AF0150 Lot number: ZZ16053 (400 mg/vial)

Details on mating procedure:

After acclimation for 11 days, animals (about 12 weeks old) meeting good health and acceptable body weight requirements (minimum 220 g) were paired with resident male rats (the same strain, source and sexually mature) [the ratio was not specified] for breeding. The gestation day 0 was defined as the day that positive evidence of mating was identified by the presence of a copulatory plug or sperm in a vaginal smear. The female rats on gestation day 0 were randomly assigned to 4 groups, 25/group (body weight of 214-270 g). All animals were individually housed through the study, except during mating with the resident male rats.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

AF0150 dose was verified by osmolality measurement.

Duration of treatment / exposure:

The male rats received daily dosing from 28 days pre-mating till sacrifice day, and the female rats were dosed daily from 14 days pre-mating till gestation day 7.

Frequency of treatment:

daily IV bolus

Details on study schedule:

After acclimation for 11 days, animals (about 12 weeks old) meeting good health and acceptable body weight requirements (minimum 220 g) were paired with resident male rats (the same strain, source and sexually mature) [the ratio was not specified] for breeding. The gestation day 0 was defined as the day that positive evidence of mating was identified by the presence of a copulatory plug or sperm in a vaginal smear. The female rats on gestation day 0 were randomly assigned to 4 groups, 25/group (body weight of 214-270 g). All animals were individually housed through the study, except during mating with the resident male rats.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

200 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

Maternal (Fo) Observations
Clinical Signs: All animals were observed twice a day for mortality and moribundity, and detailed clinical signs were recorded daily till scheduled sacrifice day (Lactation day 21).
Food consumption: Individual maternal food consumption was recorded on gestation days 0, 6, 9, 12, 15, 18 and 20, and on lactation days 1, 4, 7, 14 and 21; reported as g/animal/day and g/kg/day for each corresponding body weight change.
Body Weights: Maternal body weights were recorded on gestation days 0, 6, 9, 12, 15, 18 and 20, and on lactation day 1, 4, 7 14 and 21. Group mean body weight was calculated for each time point. Mean body weight changes were calculated for each corresponding interval and also for gestation days 0-20 and for lactation days 1-21.
Pregnancy Duration and Parturition: all surviving animals were allowed to delivery naturally and rear their offspring to weaning. The pregnancy duration was defined as days from gestation day 0 to initiation of parturition. The day of complete delivery was designated PND 0 [PND was not defined, maybe Post-Natal Day]. The number, sex and malformation of stillborn and live pups in each litter were recorded at the end of parturition.
Necropsy: all animals, including those that were scheduled for termination (on lactation day 21), unscheduled death, and those failed to delivery or had total litter loss, were subjected to necropsy. The thoracic, abdominal and pelvic cavities and contents were examined. The numbers of former implantation sites or corpora lutea (for unscheduled death) were recorded.

Litter observations:

Offspring (FI) Observations
Clinical Signs: each litter was examined daily for appearance, behavior, survival and death. Each pup received a detail physical examination on PND 1, 4, 4, 14 and 21 and weekly thereafter till necropsy. Pup sexes were determined on PND 0, 4 and 21. Eight pups per litter (4 each sex when possible) were randomly selected, weighed and sacrificed on PND 4. Liner parameters were processed as described in Table 2.

_______________________________________________________________________________________________________________

∑ (No. of viable pups per Inter / No. of implantations per litter)
Postimplantation Survival Index (% Per Litter) = ------------------------------------------------------------------------- x 100
No. of litters per group

Total no. of viable pues PND 0
Mean Live Liner Size = ------------------------------------------------------------------------- x100
No. of litters with viable pups PND 0

∑ (No. of nonviable pups at birth per litter/ No. of pups bom per litter)
Stillbirth Index (% Per Litter) = -------------------------------------------------------------------------- x100
No. of litters per group


∑ (No. of pups boni alive per littcr/ Total no. of pups boni per litter)
Live Birth Index(% Per Litter) = -------------------------------------------------------------------------- x100
No. of litters per group


Postnatal Survival Between Birth ∑ (Viable pups per liner on PND 0 or PND 4/No. of pups born per liner)
and PND 0 or PND 4 (% Per Litter)= ----------------------------------------------------------------------------- x100
No. of liners per group


Postnatal Survival for all ∑ (Viable pups per litter at end of interval N/Viable pups per liner at start of interval N) No. of liners per group
Other Intervals (% Per Litter) = ----------------------------------------------------------------------------- x100
No. of liners per group
Where N= PND 0-1, 1-4, 4-7, 7-14, 14-21 or 4-21

No. of live litters
Gestation Survival Index (%) = ------------------------------------------------------------------------------x 100
No. of pregnant females

Table 2. Calculation of litter parameters
_____________________________________________________________________________________________________________________________________________

Body Weights: individual body weight was recorded on PND 1, 4, 7, 14 and 21, and weekly thereafter tilt necropsy.
Offspring Development: Each dam and litter remained together till weaning on PND 21. Fifty pups (25 each sex) between 6-10 days old were randomly selected from each of 4 groups (saline, 50, 100, and 200 mg/kg/day AF0150) for assessment of developmental landmarks and reproductive performance. The remaining pups were sacrificed and necropsied on PND 21. The following parameters were observed in the selected pups:
1. Balanopreputial Separation: male pups were observed daily for balanopreputial separation beginning on PND 40.
2. Vaginal Perforation: female pups were observed daily for vaginal perforation beginning on PND 30.
3. Auditory Startle Test: 10 pups/sex/group on PND 21 and 60 (±5 days) received an auditory response test in the sound chamber using an automatic Auditory Startle Response System. The responses or movements of animals to the tested noise bursts were monitored by weight¬sensitive platforms on the bottom surface of the sound chamber. Mean peak amplitude (grams), latency to peak (mSec), response duration (mSec) and average response (grams) on each block (1-5) of 10 trials were recorded.
4. Motor Activity: 10 pups/sex/group on PND 13, 17, 21 and 60 (±2 days) were subjected to
motor activity test using the Animal Activity System. The animals were
placed in a clear plastic rectangular cage surrounded with series of infrared photobeams. Both fine (interrupting one or two adjacent photobeams) and ambulatory (interrupting 3 or more consecutive photobeams) motor activities were collected.
5. T-Maze Swimming Trials: learning and memory ability of pups (10 pups/sex/group) were
evaluated using the maze swimming test in a water-filled, 8-unit T-maze system. The first test was conducted between PND 20 and 24, and the second test was between PND 60 and 64. The mean number of errors and the mean escape time for each trail were considered measures of maze acquisition.
6. Reproductive Performance and Fertility of Offspring: the F1 animals at 87-91 days old were paired on a 1:1 basis within each treatment group but avoiding sibling pairing. Positive evidence of mating was confirmed by the presence of a copulatory plug or sperm in a vaginal smear, and gestation day 0 was defined as the day that positive mating was identified. Clinical signs, estrous cycles, body weights and gravid uterine weights were observed. On gestation day 20, all maternal animals were subjected to complete necropsy and fetuses (F2) were examined for malformation and variations. Female/male mating index and fertility index, post-implantation loss/liter were calculated, as described in the fertility study procedure.

Postmortem examinations (parental animals):

Maternal (F0) observations:
Necropsy: all animals, including those that were scheduled for termination (on lactation day 21), unscheduled death, and those failed to delivery or had total litter loss, were subjected to necropsy. The thoracic, abdominal and pelvic cavities and contents were examined. The numbers of former implantation sites or corpora lutea (for unscheduled death) were recorded.

Reproductive indices:

6. Reproductive Performance and Fertility of Offspring: the F1 animals at 87-91 days old were paired on a 1:1 basis within each treatment group but avoiding sibling pairing. Positive evidence of mating was confirmed by the presence of a copulatory plug or sperm in a vaginal smear, and gestation day 0 was defined as the day that positive mating was identified. Clinical signs, estrous cycles, body weights and gravid uterine weights were observed. On gestation day 20, all maternal animals were subjected to complete necropsy and fetuses (F2) were examined for malformation and variations. Female/male mating index and fertility index, post-implantation loss/liter were calculated, as described in the fertility study procedure.

Offspring viability indices:

See Table 2.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

one animal in the 200 mg/kg/day group died on gestation day 9 without a known cause. All other animals survived to the scheduled necropsy. There were no AF0150¬related toxic observations at any dose level.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

one animal in the 200 mg/kg/day group died on gestation day 9 without a known cause. All other animals survived to the scheduled necropsy

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

mean body weight and body weight gains were not significantly different between AF0150 treatment and the control during gestation and lactation, except for a slight increase (p<0.05) in body weight gain during lactation day 14-21 in the 50 and 200 mg/kg/day groups.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

there were slight decreases (p<0.05) in food consumption in the 50 mg/kg/day group during gestation days 6-9, and in the 200 mg/kg/day group during gestation days 9-12 and lactation days 7-14. All other AF0150 dose groups had no significant changes on food consumption at any time point.

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Reproductive function / performance (P0)

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Effect levels (P0)

1

Dose descriptor:

dose level:

Effect level:

ca. 200 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

mortality

Remarks on result:

other: No known cause for the death of 1/25 animal

2

Dose descriptor:

dose level:

Effect level:

ca. 50 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Slight increase in body weight gain

Remarks:

not dose-dependant

3

Dose descriptor:

dose level:

Effect level:

ca. 200 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Slight increase in body weight gain

Remarks:

not dose-dependant

4

Dose descriptor:

dose level:

Effect level:

ca. 50 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: Slight decrease during gestation days 6-9

Remarks:

not dose-dependant

5

Dose descriptor:

dose level:

Effect level:

ca. 200 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

food consumption and compound intake

Remarks on result:

other: Slight decrease during gestation days 9-12 and lactation days 7-14

Remarks:

not dose-dependant

Results: F1 generation

General toxicity (F1)

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

in the 200 mg/kg/day group there was a decrease in the live birth index (by 7% with p<0.05), postimplantation survival index (by 6.8%), mean live litter size (by 8%), live pup numbers (by 8.8%) and gestation survival index (by 5%, but without statistical significance). The number of pup loss during the postnatal period (PNDO¬21) was higher (2-fold more than in control) in the 200 mg/kg/day than the other groups, which was considered to be related to maternal exposure to AF0150.

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Description (incidence and severity):

Balanopreputal Separation and Vaginal Perforation:

All male pups had balanopreputial separation by PND 52 and all female pups had vaginal opening by PND 37 except one in the control group who did not have vaginal patency till PND 49 (due to a filamentous attachment).

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Pup Necropsy: pups that died during the postnatal period (PNDO-21) had no remarkable necropsy findings, except for one in the 50 mg/kg/day group was limited to situs inversus. For those pups scheduled for necropsy, a few pups (3, 3, 4 in the 50, 100 and 200 mg/kg/day, respectively) had dilated renal pelves. One pup in the 100 mg/kg/day group had a distended ureter.

Histopathological findings:

no effects observed

Description (incidence and severity):

Pup Necropsy: pups that died during the postnatal period (PNDO-21) had no remarkable necropsy findings, except for one in the 50 mg/kg/day group was limited to situs inversus. For those pups scheduled for necropsy, a few pups (3, 3, 4 in the 50, 100 and 200 mg/kg/day, respectively) had dilated renal pelves. One pup in the 100 mg/kg/day group had a distended ureter.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Pup Necropsy: pups that died during the postnatal period (PNDO-21) had no remarkable necropsy findings, except for one in the 50 mg/kg/day group was limited to situs inversus. For those pups scheduled for necropsy, a few pups (3, 3, 4 in the 50, 100 and 200 mg/kg/day, respectively) had dilated renal pelves. One pup in the 100 mg/kg/day group had a distended ureter.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

- Auditory Startle Test: There were no differences in auditory tests (peak amplitude, latency to peak, response duration and average response) between pups from AF0150-treated dams and pups from control dams.
- Motor Activity: Pups from the AF0150-treated groups had no differences in motor activity (total and ambulatory) as compared to the control.
- T-Maze Swimming Trial: At both testing intervals, PND 20-24 and 60-64, pups from the AF0105-treated animals had no significant changes in swimming ability, leaning and memory as compared to those in the control group.

Details on results (F1)

Offspring Observations
1. Litter and Postnatal Survival (Table 3): in the 200 mg/kg/day group there was a decrease in the live birth index (by 7% with p<0.05), postimplantation survival index (by 6.8%), mean live litter size (by 8%), live pup numbers (by 8.8%) and gestation survival index (by 5%, but without statistical significance). The number of pup loss during the postnatal period (PNDO¬21) was higher (2-fold more than in control) in the 200 mg/kg/day than the other groups, which was considered to be related to maternal exposure to AF0150.
2. Pup Body Weight: there were no significant changes on pup body weights during the observation period (PNDO-21).

Pup Necropsy: pups that died during the postnatal period (PNDO-21) had no remarkable necropsy findings, except for one in the 50 mg/kg/day group was limited to situs inversus. For those pups scheduled for necropsy, a few pups (3, 3, 4 in the 50, 100 and 200 mg/kg/day, respectively) had dilated renal pelves. One pup in the 100 mg/kg/day group had a distended ureter.
Table 3. Gestation Outcomes and Postnatal Survival
Observations AF0150 (mg/kg/day)
0 50 100 200
Number of Dams 23 25 25 23
Pregnancy Rate (%) 92 100 100 96
Gestation Days 21.8±0.42 21.7±0.54 21.8±0.37 21.7±0.45
Gestation Survival Index (% Live Litter) 100 100 100 95.7
Post-implantation Survival Index (% Per Litter) 94.2±6.73 95.7±4.91 92.7±10.16 87.6±21.58
Stillbirth Index (% Per Litter) 0.3±1.48 0.2±1.12 2.4±9.39 7.7±21.17
Live Birth Index (PND 0) 99.7±1.49 99.8±1.11 97.6±9.38 92.3±21.17*
Live Litter Size (PND 0) 15.1±2.17 15.4±1.85 15.3±2.08 13.9±3.65
Live Pups No. 317 379 382 289
Total Postnatal Death (PNDO-21) 26 4 13 38
Postnatal Missing and cannibalized (PNDO-21) 9 3 0 26
* n<0.05. as compared to the control croup (0 mg/kg/day AF0150).

Offspring (F1) Development
1. Balanopreputal Separation and Vaginal Perforation: All male pups had balanopreputial separation by PND 52 and all female pups had vaginal opening by PND 37 except one in the control group who did not have vaginal patency till PND 49 (due to a filamentous attachment).
2. Auditory Startle Test: There were no differences in auditory tests (peak amplitude, latency to peak, response duration and average response) between pups from AF0150-treated dams and pups from control dams.
3. Motor Activity: Pups from the AF0150-treated groups had no differences in motor activity (total and ambulatory) as compared to the control.
4. T-Maze Swimming Trial: At both testing intervals, PND 20-24 and 60-64, pups from the
AF0105-treated animals had no significant changes in swimming ability, leaning and memory as compared to those in the control group.


Reproductive performance and Fertility of Offspring: A routine fertility procedure was followed to test the reproductive function of both male and female pups.
i. No remarkable clinical signs and body weight changes were observed in both male and
female offspring from all groups before, during and alter gestation.
ii. There were no significant differences in estrous cycle, fertility index and mating index in
both sexes between AF0150- and control groups.
iii. Gestation Day 20 laparohysterectomy, fetal (F2) development and morphology were not
remarkable in any group, except for a filamentous tail noted in one fetus from the 200 mg/kg/day group and a short tail in one fetus from the 50 mg/kg/day group. These malformation were considered to be spontaneous based on the historical control data of this laboratory
iv. At necropsy on gestation day 20, one female from the 50 mg/kg/day group and 2 females from the 200 mg/kg/day group had dilated renal pelves, distended ureters, calculi in both kidneys, and another two females from the 200 mg/kg/days had a cystic ovary. One male from the control, 2 males from the 50 mg/kg/day group and 1 male from the 200 mg/kg/day group had small and/or soft testes.

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

2

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

not determinable due to absence of adverse toxic effects

3

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

>= 50 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: Dilated renal pelvis

4

Key result

Dose descriptor:

NOAEL

Generation:

F1

Based on:

test mat.

Sex:

male/female

Basis for effect level:

sexual maturation

Remarks on result:

not determinable due to absence of adverse toxic effects

5

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 200 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: Auditory Startle test

Remarks on result:

not determinable due to absence of adverse toxic effects

6

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 200 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: Motor activity

Remarks on result:

not determinable due to absence of adverse toxic effects

7

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: T-Maze swimming trial

Remarks on result:

not determinable due to absence of adverse toxic effects

8

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Estrous cycle, fertility index, mating index

Remarks on result:

not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Table 3.Gestation Outcomes and Postnatal Survival

Observations	AF0150 (mg/kg/day)
	0	50	100	200
Number of Dams	23	25	25	23
Pregnancy Rate (%)	92	100	100	96
Gestation Days	21.8±0.42	21.7±0.54	21.8±0.37	21.7±0.45
Gestation Survival Index (% Live Litter)	100	100	100	95.7
Post-implantation Survival Index (^(Vo Per Litter)	94.2±6.73	95.7±4.91	92.7±10.16	87.6±21.58
Stillbirth Index (% Per Litter)	0.3±1.48	0.2±1.12	2.4±9.39	7.7±21.17
Live Birth Index (PND 0)	99.7±1.49	99.8±1.11	97.6±9.38	92.3±21.17*
Live Litter Size (PND 0)	15.1±2.17	15.4±1.85	15.3±2.08	13.9±3.65
Live Pups No.	317	379	382	289
Total Postnatal Death (PNDO-21)	26	4	13	38
Postnatal Missing and cannibalized (PNDO-21)	9	3	0	26
* n<0.05. as compared to the control group (0 mg/kg/day AF0150).

Applicant's summary and conclusion

Conclusions:

This prenatal/postnatal study was conducted in rats given daily IV injections of AF0150 at 0 (saline), 50, 100 and 200 mg/kg/day from gestation day 6 through lactation day 20. Observations included maternal toxicity (clinical signs, body weight, food consumption and necropsy), gestation, parturition, lactation, neonate/offspring survival and development (growth, behavior, motor activity, reproductive performance and fertility).
AF0150 did not significantly affect maternal toxicity, pregnancy rate and gestation duration at any dose level.
However, at the dose of 200 mg/kg/day, AF0150 decreased the live birth index (by 7% with p<0.05), postimplantation survival index (by 6.8%), mean live litter size (by 8%), live pup numbers (by 8.8%) and gestation survival index (by 5%, without statistical significance). AF0150 increased stillbirths from 0.3% (in the control group) to 7.7% (in the 200 mg/kg/day group) per liner (about 25-fold higher). There was also approximately 2-fold increase in total postnatal death and postnatal missing/cannibalized during the postnatal days 0-21 in the 200 mg/kg/day group. The NOAEL for neonate toxicity was 100 mg/kg/day (HED = 16 mg/kg/day and HDM = 130-fold).
The offspring from the AF0150-treated animals had no differences in physical and functional development as well as in behavior from those from the control group. Fertility study on the offspring from the AF0150-treated groups showed no significant effects on fertility and mating indices in either sex, nor on fetal development and morphology, as compared to the offspring from the control group.
This study suggests that exposure to AF0150 at the high dose (200 mg/kg/day) during pregnancy and lactation increases the possibility of pre- and postnatal toxicity.

However, as AF150 is a tetradecafluorohexane in a galenic dramatically increasing the biodisponibility, as AF150 was administered by iv route, hence dramatically increasing systemic exposure, as tetradecafluorohexane administered by oral route is absorbed 10% max and then rapidly eliminated in exhaled air, and as the effects observed with AF150 are unspecific and limited to high dose group, these effect should be considered not relevant for tetradecafluorohexane not embeded in lipid spheres, administered by oral route.