Tetradecafluorohexane

Toxicity to reproduction: other studies

Administrative data

Endpoint:

toxicity to reproduction: other studies

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

february 26 - april11, 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:

1997

Report date:

1999

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD TG 421

Deviations:

yes

Remarks:

Sacrifice on gestation day 13

Principles of method if other than guideline:

Specific Aim
To assess effects of AF0150 on fertility and early embryonic development in rats (Segment I). Methods
Animal Preparation: Crl:CD(SD)BR rats (100 each sex, 45 days old) were obtained from
Standard procedures were followed for housing, handling, feeding and care of the animais. After acclimation and quarantine for 13 days (for males) and 17 days (for females, the first day of smearing for estrus), animais (namely Fo generation) meeting good health and acceptable body weight requirements were randomly assigned into 4 groups (25 rats/sex/group) and received designed treatments, as seen in Table 1.
Group AF0150 Dose*
(mg/kg/day) Number of Rats IV Volume
(ml/kg/day)
Male Female
1 (Control) (Saline) 25 25 5.0
2 (AF0150) 50 25 25 1.25
3 (AF0150) 100 25 25 2.5
4 (AF0150) 200 25 25 5.0

Animais were dosed from pre-mating (28 days) till the sacrifice day for male rats, and from pre-mating (14 days) till the gestation Day 7 and sacrificed on gestation day 13. * AF0150 was reconstituted in SWFI to final concentration of 40 mg/ml.
AF0150 Preparation and Administration: AF0150 (400 mg fill) was reconstituted with 10 ml SWFI to a final concentration of 40 mg/kg, and used within 30 minutes after reconstitution. The solutions were not used if they turned clear and could not be restored (by shaking) to an opaque appearance. 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). The AF0150 dosages were 50, 100 and 200 mg/kg/day for assigned groups, and 5 ml/kg/day of saline for control group (Table 1). 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. Individual dosages were based on the mort recent body weights. All animais were treated at approximately the same time each day.
Observations: the animais were observed twice a day for mortality and moribundity, and detailed clinical signs were recorded daily. Body weights were measured twice weekly throughout the dosing period (up to day 55). The females with evidence of mating were weighed on gestation days 0, 3, 7, 10 and 13. Food consumption was recorded twice weekly or on the gestation days for body weight measurement, expressed as g/animal/day and g/kg/day.
Estrous Cycles (for Fo females): Vaginal smears (for determining the stage of estrous) were conducted daily for each Fo female for 10 days pre-dosing and throughout the 14-day pre-mating/dosing period. Once the animais were paired, smearing was continued until evidence of copulation was observed, or until termination of the mating period.
Breeding Procedures: Following a 28-day dosing period, Fo males were paired with Fo females (which had undergone a 14-day dosing) from the same treatment group. The animais were paired on a 1:1 basis within each treatment group. Each mating set was examined daily. Positive evidence of mating was confirmed by the presence of sperm in a vaginal smear or a vaginal copulatory plug. The day with evidence of mating was identified as the gestation day 0. If evidence of copulation was not detected after 10 days of pairing, a female was placed with a proven male from the same treatment group for an additional 5 days. Pre-coitai intervals were calculated according to a 12-hour dark cycle counted for one paired day. Mating and fertility indices were calculated according to the equations described in Table 2.

Table 2. Calculation of Mating and Fertility Indices

Male (Female) Mating Index (%) Numbers of Males (Females) with Positive Mating
------------------------------------------------------------
Total Numbers of Males (Females) used for Mating

Male Fertility Index (%) — x 100 Numbers of Males Siring at least 1 Liter
----------------------------------------------
Total Numbers of Males Used for Mating

Numbers of Females with Confirmed Pregnancy
Female Fertility Index (%) — ---------------------------------------------------------- x 100
Total Numbers of Females used for Mating

Gestation Day 13 Uterine Examination: females with evidence of mating were sacrificed on gestation day 13. The number of corpora lutea on each ovary and the number/location of all embryos, early resorptions and implantation sites in the uterus were recorded. Additionally, the thoracic and abdominal cavities of females with evidence of mating were examined. Viability of the embryos was determined with a dissecting stereomicroscope. Data were processed with the equations described in Table 3.

Table 3. Gestation data process

Group Mean Litter Basis:
Numbers of Dead Embryos, Resorptions (Early/Late)!Group
Post-Implantation Loss/Litter = ----------------------------------------------------------------------
Numbers of Gravid Females/Group
Proportional Litter Basis:
Total Post-Implantation Loss/Litter/Group (%)
Summation per Group (%)= -----------------------------------------------------
Numbers of Litters/Group

Numbers of Dead Embryos, Resorptions/Litter
Total Post-Implantation Loss/Litter/Group (%)= ------------------------------------------------------- x 100
Numbers of Implantation Sites/Litter



Spermatogenetic Endpoint Evaluations: male rats was sacrificed and necropsied after the female necropsy, and spermatogenetic endpoints were evaluated, including testicular and epididymal sperm numbers, sperm production rate, sperm motility and sperm morphology.
Macroscopic Examination: ail animals were subjected to a complete necropsy at the scheduled sacrifice (on gestation day 13). The necropsy included examination of the externat surface, ail orifices, the cranial cavity, the externat surface of the brain and spinal cord, and the thoracic, abdominal and pelvic cavities. The absolute and relative weight (to final body weight) of testes, epididymides (total and cauda), ovaries, brain and pituitary gland were determined.
Microscopic Examination: at necropsy, the following tissues/organs were collected and preserved for possible future histopathological examination:

Coagulating gland
Ovaries and oviduct (2)
Pituitary
Prostate
Seminal vesicies (2)
Right testis with epididymis (1)a
and vas deferens
Litem with vagina
All gross lesionsb

a.The right testis and epididymis were fixed in Bouin's solution.
b.Representative sections of corresponding organs from control animais were preserved for comparison.

GLP compliance:

yes

Type of method:

in vivo

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: CD(SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

Crl:CD(SD)BR rats (100 each sex, 45 days old) were obtained from
Standard procedures were followed for housing, handling, feeding and care of the animais. After acclimation and quarantine for 13 days (for males) and 17 days (for females, the first day of smearing for estrus), animais (namely Fo generation) meeting good health and acceptable body weight requirements were randomly assigned into 4 groups (25 rats/sex/group) and received designed treatments, as seen in Table 1.
Group AF0150 Dose*
(mg/kg/day) Number of Rats IV Volume
(ml/kg/day)
Male Female
1 (Control) (Saline) 25 25 5.0
2 (AF0150) 50 25 25 1.25
3 (AF0150) 100 25 25 2.5
4 (AF0150) 200 25 25 5.0

Animais were dosed from pre-mating (28 days) till the sacrifice day for male rats, and from pre-mating (14 days) till the gestation Day 7 and sacrificed on gestation day 13. * AF0150 was reconstituted in SWFI to final concentration of 40 mg/ml.

Administration / exposure

Route of administration:

intravenous

Vehicle:

water

Details on exposure:

AF0150 Lot number: ZZ16051, ZZ16052, ZZ16053 (400 mg/vial) 50

Analytical verification of doses or concentrations:

not specified

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. Individual dosages were based on the mort recent body weights. All animals were treated at approximately the same time each day.

Frequency of treatment:

Daily IV bolus

Duration of test:

In-life phase: February 26 — April 11, 1997

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:

50 (25 per sex)

Control animals:

yes, concurrent vehicle

Details on study design:

Group AF0150 Dose*
(mg/kg/day) Number of Rats IV Volume
(ml/kg/day)
Male Female
1 (Control) (Saline) 25 25 5.0
2 (AF0150) 50 25 25 1.25
3 (AF0150) 100 25 25 2.5
4 (AF0150) 200 25 25 5.0

Animals were dosed from pre-mating (28 days) till the sacrifice day for male rats, and from pre-mating (14 days) till the gestation Day 7 and sacrificed on gestation day 13. * AF0150 was reconstituted in SWFI to final concentration of 40 mg/ml.

Results and discussion

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

>= 200 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Reproductive performance

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

there were no significant differences in mating index, fertility index and pre-coital intervals between the AF0150-treated and the control groups. AF0150 at all doses had no effects on the regularity and duration of the estrous cycle.

2

Key result

Dose descriptor:

NOAEL

Effect level:

>= 200

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Reproductive performance

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

There were no significant AF0150-related effects on male reproductive performance at all dose levels tested, except for a slight decrease (by 5%) in fertility index noted in the males from the 200 mg/kg group without statistical significance as compared to the control group.

3

Dose descriptor:

NOAEL

Effect level:

>= 200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Remarks on result:

not determinable due to absence of adverse toxic effects

4

Dose descriptor:

NOAEL

Effect level:

ca. 50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

food consumption and compound intake

Remarks on result:

other: Slight decrease in food consumption (5-7%)

Remarks:

Male: Food consumption slightly decreased (by 5%, p<0.05) in the 100 and 200 mg/kg groups at the end of study (Days 51-55), and no remarkable changes were observed in other groups and at any time points. Female: During the pre-mating period, food consumption (g/animal/day and g/kg/day) in all AF0150 dose groups at most time points were not different from the control group, except that a slight but statistically significant decrease (by 6-7%) in the 100 mg/kg/day on Days 16-20 was noted. During the gestation period, there were no differences in food consumption at All AF0150 doses and time points as compared to the control group.

5

Dose descriptor:

NOAEL

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

6

Dose descriptor:

NOAEL

Effect level:

>= 200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Remarks on result:

not determinable due to absence of adverse toxic effects

7

Dose descriptor:

NOAEL

Effect level:

ca. 100 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Mean epididymal sperm number

8

Dose descriptor:

NOAEL

Effect level:

>= 200 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Testicular Sperm Count

Remarks on result:

not determinable due to absence of adverse toxic effects

9

Dose descriptor:

NOAEL

Effect level:

>= 200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Macrospopic examination

Remarks on result:

not determinable due to absence of adverse toxic effects

10

Dose descriptor:

NOAEL

Effect level:

>= 200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

The organ weights (absolute and relative) of brain, testis (right and left), epididymis (right and left) and pituitary were not different between the treated and control groups.

11

Dose descriptor:

NOAEL

Effect level:

>= 200 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Gestation day 13 uterine examination

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

AF0150 at all doses had no effects on pre- and post-implantation losses, numbers of viable embryos, corpora lutea and implantation sites, as compared with control.

Observed effects

The effects of AF0150 on fertility were assessed in male and female rats. AF0150, at the doses of 50, 100 and 200 mg/kg/day, was given to the animals from 2 (female) and 4 (male) weeks pre-mating till post-mating sacrifice day (male) and gestation day 7 (female). Slight decreases in male fertility (by 5%, not statistically significant) and mean epididymal sperm numbers (by 15%, p<0.05) were observed in the high dose groups (200 mg/kg/day), with NOAELs of 100 mg/kg/day (HED: 16.2 mg/kg/day and HDM: 130-fold of PCD) for both findings.
There were no significant differences in the other observations of reproductive performance (mating index, fertility index, pre-coital interval, and female estrous cycle) and male spermatogenesis (testicular sperm coures, sperm production rate, sperm motility and morphology) between AF0150-treated and control groups. The female necropsy on gestation day 13 showed that AF0150 had no significant effects on pre- and post-implantation losses, number of viable embryos, implantation sites and corpora lutea.
However, at the high AF0150 dose of 200 mg/kg/day, no maternai toxicity was noted, including changes on body weights, organ weights (brain, testis, ovary and pituitary) and food consumption. Minimal toxicity is generally expected in the high-dose group for appropriate dose selection.

Any other information on results incl. tables

Male Reproductive Observations

Clinical Signs, Body Weight and Food Consumption:All male rats survived to the schedule sacrifice. No AF0150-related toxic signs were noted at the daily observations. Transient changes (increase or decrease) in body weight gain were noted in some AF0150-treated animals at certain time points, but without dose-dependency. For example, body weight gains in all males on Days 27 and 48 in both control and AF0150 groups (vol 025, Table 7, p230) were much lower than those at other time points. No appropriate explanation was provided. Food consumption slightly decreased (by 5%, p<0.05) in the 100 and 200 mg/kg groups at the end of study (Days 51-55), and no remarkable changes were observed in other groups and at any time points.

Reproductive Performance (Table 4):There were no significant AF0150-related effects on male reproductive performance at all dose levels tested, except for a slight decrease (by 5%) in fertility index noted in the males from the 200 mg/kg group without statistical significance as compared to the control group.

Table 4.Male Reproductive Performance
Parameter	AF0150 (mg/kg/day),n=25
	0	50	100	200
Mating Index(%)	96	96	96	95.8
Fertility Index(%)	92	91.7	100	87.5*
Pre-coital Interval days, Mean * SD)	2.3 ± 1.27	2.7 ± 1.33	2.9 ± 1.02	2.9 ± 2.45

 

* The decrease was not statistically significant as compare to the control (92%) with Chi-square test.

Spermatogenetic Endpoint Evaluation (Table 5):Mean epididymal sperm number in the 200 mg/kg/day group decreased by 14 % with statistical significance. The other spermatogenic parameters including mean testicular sperm numbers, sperm production rate, sperm motility and morphology were not significantly different between AF0150 (at All dose levels) and control groups.

Table 5.Testicular and Epididymal Sperm Counts

Organs	AF0150 (mg/kg/day),n=25
	0	50	100	200
Left Testis ean t SD)	88.3 ± 15.9	95.9 ± 16.3	98.6 ± 13.6*	83.3 ± 13.6
Left Epididymis ean *SD	514.5 ± 93.2	464.4 ± 85.7	479.8 ± 142.7	443.6 ± 73.5*

 

* Compared to the control (0 mg/kg/day, saline) group with Dunnett's Test, p<0.05

Macroscopic Examination:No remarkable AF0150-related macroscopic findings were noted at necropsy. The organ weights (absolute and relative) of brain, testis (right and left), epididymis (right and left) and pituitary were not different between the treated and control groups.

Female Reproductive Observations

Clinical signs:all female rats survived to the schedule necropsy. No AF0150-related toxic signs were noted at the daily observations.

Body Weight:During the pre-mating period, mean body weight and body weight gains at all dose levels and at most time points were not different from the control group, except that body weight gains increased in the 200 mg/kg/day during Days 1346 and in 50 and 100 mg/kg/day groups during days 44-48 (with statistical significance). During the gestation period, AF0150 at all doses did not change body weight and body weight gain as compared with the control group.

Food Consumption:During the pre-mating period, food consumption (g/animal/day and g/kg/day) in all AF0150 dose groups at most time points were not different from the control group, except that a slight but statistically significant decrease (by 6-7%) in the 100 mg/kg/day on Days 16-20 was noted. During the gestation period, there were no differences in food consumption at All AF0150 doses and time points as compared to the control group.

Reproductive Performance:there were no significant differences in mating index, fertility index and pre-coital intervals between the AF0150-treated and the control groups. AF0150 at all doses had no effects on the regularity and duration of the estrous cycle.

Gestation Day 13 Uterine Examination:AF0150 at all doses had no effects on pre- and post-implantation losses, numbers of viable embryos, corpora lutea and implantation sites, as compared with control.

Macroscopic Examination:No remarkable findings at the scheduled and unscheduled necropsy were observed in both AF0150-treated and control groups. Some incidental findings were noted, such as dilated renal pelvis (1/24 rats at 50 mg/kg/day), dark red lung (1/24 rats at 50 mg/kg/day), hemorrhagic/thickened right parietal skull and a cyst in the lefl oviduct (1/24 rats at 200 mg/kg/day). AF0150 at all doses had no adverse effects on the organ weights (absolute and relative) of brain, ovaries and pituitary gland.

Applicant's summary and conclusion

Conclusions:

The overall conclusion of the present study is the lack of observed adverse effect on reproduction-specific endpoints.
Appart from epidydime sperm count, which had no consequence on reproductive performance, only slight non-specific effects were observed at the high dose, and a few not dose-dependant minor effeccts were observed.

Executive summary:

The effects of AF0150 on fertility were assessed in male and female rats. AF0150, at the doses of 50, 100 and 200 mg/kg/day, was given to the animals from 2 (female) and 4 (male) weeks pre-mating till post-mating sacrifice day (male) and gestation day 7 (female). Slight decreases in male fertility (by 5%, not statistically significant) and mean epididymal sperm numbers (by 15%, p<0.05) were observed in the high dose groups (200 mg/kg/day), with NOAELs of 100 mg/kg/day (HED: 16.2 mg/kg/day and HDM: 130-fold of PCD) for both findings.

There were no significant differences in the other observations of reproductive performance (mating index, fertility index, pre-coital interval, and female estrous cycle) and male spermatogenesis (testicular sperm coures, sperm production rate, sperm motility and morphology) between AF0150-treated and control groups. The female necropsy on gestation day 13 showed that AF0150 had no significant effects on pre- and post-implantation losses, number of viable embryos, implantation sites and corpora lutea.

However, at the high AF0150 dose of 200 mg/kg/day, no maternai toxicity was noted, including changes on body weights, organ weights (brain, testis, ovary and pituitary) and food consumption. Minimal toxicity is generally expected in the high-dose group for appropriate dose selection.