Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August - September 1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Near guidleline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1981

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Only seven animals used per group. Only 2 exposure groups
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Substance: Perfluorobutylethene - PFBE (3,3,4,4,5,5,6,6,6-nonafluorohexene)
Physical form: Colorless liquid
Date received: 6 July 1981
Amount received:
Container #1: 24.900 Kg
Container #2 : 26.475 Kg
Container #3: 26.650 Kg
Container #4: 26.500 Kg

Test animals

Species:
rat
Strain:
other: CRl: CD® (SD) (BR)
Details on test animals and environmental conditions:
59 female CD® (Sprague-Dawley derived) rats were supplied by Charles River Breeding Laboratories Inc. Wilmington, Massachusetts. The animals were 56 days old when received on 30 July 1980. 21 females were put on test, being mated at 69 days of age with males from an in-house mating colony fo the same strain of rat.

Females were housed individually (except during overnight mating intervals) in stainless steel, suspended cages with wire mesh floors. The animals were fed Certified Rodent Chow #502 ad libitum during the non-exposure intervals. Tap water was available ad libitum via an automatic watering system during the non-exposure intervals. During the exposure periods animals did not have access to either food or water.

Temperature was maintained at 21 - 25 °C, humitdy at 42 -70%, and photo period 12 hours light/dark cycle.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
Test material delivery:
The test material was placed in a 50 milliliter (ml) glass syringe mounted on 1 Sage syringe pump (Model #352) for the low exposure (Group II). For the high exposure(Group III), approximately 1600 mls of PFBE was placed in a 2000 ml flask connected to an FMI Lab Pump(Model G-20), From these reservoirs, the test material was metered to a 4-neck round-bottom flask that was maintained at a temperature of 45°C using a water bath. Housellne dry air, at a flow rate of 7.0 liters per minute (lpm), was directed into the flask. The resultant vapour-laden airstream was delivered Into the exposure chamber.

Chamber operation:
The animals were exposed in a glass chamber with a volume of 19.7 litres. The average air flow rate through the chamber was 7.0 l/min. This flow was calculated to provide one complete air change every 2.8 minutes and a 99% equilibrium time of 12.9 minutes.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations were determined in the chambers, including control, every half hour using a GOW-MAC Model #750 gas chromatograph. A six foot 1/8" chromosorb 102 column at a temperature of 160°C was employed for the chromatographic separation and determination. Chamber concentrations were determined by comparing the integrated GC responses obtained with primary gas standards. The GC sampling sequence was Groups I, II, III, I etc. However, because of the carry-over of PFBE in the sampling lines from high dose to control, this sequence was reversed on Day 2 and on Day 6 the sampling time was increased from 8 to 9 minutes.
Chamber oxygen was monitored daily (4 times per day for the first two days and twice daily thereafter) using an MSQ Portable Oxygen Indicatory (Type E).
Chamber air temperatures were recorded hourly during the exposures.
Details on mating procedure:
Females selected for mating were placed with male rats nightly in a 1:1 ratio. Vaginal smears were taken early in the morning and females were considered mated if sperm were observed microscopically in the vaginal rinse. The day when evidence of mating was observed was designated Day 1 of gestation.
Duration of treatment / exposure:
Mated females were exposed daily for 6 hours/day from Day 6 to Day 15 of gestation.
Duration of test:
Day 6 to Day 15 of gestation represented a 10 day treatment period.
No. of animals per sex per dose:
7 pregnant females
Control animals:
yes, sham-exposed

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily females were observed for obvious signs of pharmacologic or toxicologic effects and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: each female was given a detailed physical examination on Days 1, 6, 9, 12, 15, 18 and 21 of gestation. During the treatment period these examinations were performed in the afternoon following exposure.

BODY WEIGHT: Yes
- Time schedule for examinations: Females were weighed on Days 1, 6, 9, 12, 15, 18 and 21 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
Food consumption was recorded during the exposure (days 6 - 9, 9-13 and 13-16) and post exposure (days 16-21) periods. Food spillage was weighed and recorded during the same intervals that feeder jars were weighed.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21 of gestation
- Organs examined: uterus, ovaries, liver

Ovaries and uterine content:
Each ovary was dissected free of the uterus, cleaned of connective tissue and evaluated for the presence and number of corpora lutea.

Uterus - the number and relative location of the following were recorded for each uterine horn:

Live fetuses

Dead fetuses (dead fetus with no visible degeneration)

Late resorptions (recognizable dead fetus undergoing degeneration regardless of size)

Early resorptions (evidence of implantation but no recognizable fetus)

Implantation sites (represents the sum total of fetuses (live and dead) and resorption sites (early and late)

Once fetuses were removed from the uterus and all implantation sites identified, the placental/extra embryonic tissues were removed and uterus re-weighed (empty weight).
Fetal examinations:
Upon removal from the uterus, each fetus was given a gross examination for malformations, weighed, sexed and tagged for future identification.

Fetal soft tissue examination:
Approximately one half of the fetuses in each litter were evaluated for soft tissue malformations by the Staple micordissection technique.
Fetuses so processed were decapitated and heads fixed in Bouin's solution to be processed by a razor blade slicing technique. At completion of the soft tissue examination, the decapitated, eviscerated specimens were processed for the staining of the skeletal structures using an Alizarian Red S staining procedure. Stained fetal specimens were subsequently evaluated under a dissecting microscope for malformations and ossification variations.

Fetal skeletal examinations:
The remaining fetuses in each litter were eviscerated (external sexing data confirmed by internal inspection of the gonads) and processed for staining of the skeletal structures using an Alizarian Red S Staining Procedure. Stained fetal specimens were evaluated under a dissecting microscope for skeletal malformations and ossification variations.
Statistics:
See 'Any other information on materials and methods'
Historical control data:
Historically, the incidence of ureter observations (distended or tortuous ureter) is 8.7% in the term rat in this laboratory (representing 1067 fetuses from 100 litters).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
No treatment-related effect was evident in body weight or food consumption data for the low-dose group. At the high-dose level, mean body weight gain for the day 6-16 gestation interval (exposure period) was significantly lower than control. Mean food consumption data for the hlgh-dose group was lower than control for the exposure (day 6 -16) and post-exposure (day 16-21) intervals; however, only during the exposure period was the difference from control data statistically significant.
No mortality or adverse maternal efftects were seen in physical in-life examination data, liver weight data or gross postmortem examination data. Likewise, corpora lutea and uterine implantation data, fetal weight and sexing data and ossification variation data did not appear to be adversely affected by treatment.

Effect levels (maternal animals)

Dose descriptor:
NOAEC
Effect level:
70 000 ppm
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
organ weights and organ / body weight ratios
gross pathology
pre and post implantation loss

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
See below 'Any other information on results'

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEC
Effect level:
> 70 000 ppm
Based on:
test mat.
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEC
Effect level:
> 70 000 ppm
Based on:
test mat.
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Exposure Atmosphere Analytical Data

The mean nominal exposure chamber concentrations were 1050 and 69,600 for the groups exposed to be PFBE (Groups II & III). The cumulative analytical concentrations of PFBE were 994 and 70,700 ppm for Groups II and II respectively. The differences between nominal and analytical concentrations are within the limits of experimental error. For the first two days of exposure, concentrations of approximately 25ppm were observed for samples taken from the control chamber. This was attributed to carry-over in the sampling lines from high dose to control. This problem was eliminated by reversing the sampling sequence and increasing the sampling time. During the remainder of the control exposures, peak areas observed were either very small or not present at all. The oxygen concentration in the exposure chambers ranged from 19.0 to 20.8%.

Fetal body weight and sex distribution data

Mean fetal body weight were considered comparable between the control and treated groups. No stunted fetuses were recovered either in the control or treated groups. Likewise, no fetuses were calculated as being stunted in respects to littermates in these same groups.

Fetal sex distribution data revealed a preponderance of males in each of the treated groups with an increase in the number of females in the control group. The differences in sex distribution are attributed to normal biological variability noted with this parameter and the relatively small group sizes involved.

No treatment related effect was evident in fetal body weight or fetal sexing data.

 

Fetal external examination data

No external malformations were seen in the 84 low-dose fetuses (seven litters), or 85 high-dose fetuses (seven litters).

In the control group, one fetus had a darkened, raised area on its back (thoracic region). No other external malformations were seen in the 90 remaining fetuses for the control group (seven litters).

Fetal soft tissue examination data

The most common observations noted during the soft tissue evaluations involved distention and/or the tortuous condition of the ureter (s). These types of observation were seen in 2/48 controls (4.2%), 6/43 low-dose fetuses, and 2/44 high-dose fetuses (4.5%). The significance of these types of observations is unclear. In these same fetuses with ureter observations, the bladders were noted as filled (indicating a patency between urteter and bladder) and no kidney malformations (e.g. severely distended renal pelvis, absence of renal papillae) were seen. These types of ureter observations (i.e. distended , tortuous condition) are seen frequently in the term rat fetuses; historically, the incidence of such ureter observations is 8.7% (representing 1067 fetuses from 100 litters). Since no dose relationship was apparent in the incidence of these ureter observations, no treatment-related effect was indicated.

With the exception of the ureter observations, the only other observation noted during the soft tissue evaluations was a small, dark, discolored area on the medium lobe of the liver. This observation was seen in one low-dose fetus.

Thus, no treatment-related effect on the fetus was evident from data collected during the soft tissue evaluation.

Ossification variation data

Ossification variations were seen in 87/91 control fetuses, 75/84 low-dose fetuses and 77/85 high-dose fetuses, and incidence of 95.6%, 89.3% and 90.6% respectively. These data were comparable between control and each treated group.

The types and incidences of ossification variations seen were similar between the control and treated groups. No treatment-related effect was evident in these data.

Fetal skeletal examination data

The incidence of skeletal malformations, both on a per fetus and a per litter basis, was comparable between the control and treated groups.

In the control group, one fetus had an angulated/shortened rib defect. No other skeletal malformations were seen in this group.

Fused sternebrae were seen in two treated group fetuses. In the low-dose group, one fetus from the litter of female No. 2580 had a fusion between the first and second sternebrae. The fused sternebrae seen in one-high dose fetus from the litter of female No. 3578, involved the second through the fifth sternebrae. A mid-line cleft was also evident through this fused segment of the sternum.

The sternebral fusion defects seen in a single fetus from each of the treated groups were dissimilar in respects to the sternebrae involved and were not considered to represent a treatment-related effect.

 

During the external, soft tissue and skeletal evaluations, malformations were seen in 4/91 control fetuses (4.4%), 8/84 low-dose fetuses (9.5%) and 3/85 high-dose fetuses (3.5%). The mean percentages of malformed fetuses per litter for the control, low- and high-dose groups were 4.3%, 9.8% and 3.3% respectively. Though these values are increased in the low-dose group, differences from control were not statistically significant. Likewise, no dose-relationship was evident in these values.

It was concluded that exposure to PFBE by whole-body inhalation at levels of 1000 and 70,000 ppm during the Days 6 through 15 of gestation did not appear to be embryotoxic or teratogenic in the seven pregnant rats evaluated per dose level.

Applicant's summary and conclusion

Conclusions:
No evidence of embryotoxicity or treatment-related malformations were seen in the offspring of rats exposed during days 6-15 of gestation to levels of PFBE (3,3,4,4,5,5,6,6,6-nonafluorohexene) of 1,000 or 70,000 ppm.
Executive summary:

This pilot study was conducted to evaluate the embryotoxic and/or teratogenic potential of PFBE (3,3,4,4,5,5,6,6,6-nonafluorohexene) in the pregnant rat. Mated CD® female rats (seven female/group) were exposed by inhalation to PFBE at levels of 1,000 and 70,000 ppm. Animals were exposed for six hours/day during the day 6-15 of gestation. Included in the study was a sham-air, chamber-exposed control group (seven mated females), All females were sacrificed on Day 21 of gestation and fetuses recovered at this time were weighed, sexed and evaluated for external, soft tissue and/or skeletal malformations. Fetuses processed for skeletal evaluation were also examined for the presence of ossification variations.

No maternal mortality was encountered during the study and all females, control and treated groups were pregnant.

No treatment-related effect was evident in body weight or food consumption data for the low-dose group. At the high-dose level, rnean body weight gain for the Day 6-16 gestation interval (exposure period) was significantly lower than control. Mean food consumption data for the hlgh-dose group was lower than control for the exposure (Day 6 -16) and post-exposure (Day 16-21) intervals; however, only during the exposure period was the difference from control data statistically significant.

No adverse maternal effects were seen in physical in-life examination data, liver weight data or gross post-mortem examination data. Likewise, corpora lutea and uterine implantation data, fetal weight and sexing data and ossification variation data did not appear to be adversely affected by treatment.

No increase in malformation rate was seen in the treated groups during the fetal external or skeletal evaluations. During the soft tissue evaluations, a slight increase in the incidence of fetuses with malformations was seen only in the low dose group.The most common observation noted during this evaluation involved distention and/or tortuous condition of the ureter. Similar type observations were seen in the concurrent control group with a lower incidence, and are noted frequently in our historical data for this strain of rat. In that a similar response was not noted at the higher exposure level, no treatment-related effect was indicated.