1,1,2,2,3,3,4-heptafluorocyclopentane

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 1997-10-20 to 1997-11-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

1981

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

430-710-1

EC Name:

-

Cas Number:

15290-77-4

Molecular formula:

C5H3F7

IUPAC Name:

1,1,2,2,3,3,4-heptafluorocyclopentane

Specific details on test material used for the study:

Batch No.: 9708-1A
Purity: 98.92 %

Test animals

Species:

rat

Strain:

other: Crl: CD BR VAF/Plus strain

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited
- Age at study initiation: 8-11 weeks
- Weight at study initiation: 203-226 g
- Fasting period before study: not fasted
- Housing: housed, 5 per cage, in suspended cages with stainless steel mesh on top, front, back and floor and stainless steel side panels. Each cage was 35 cm wide, 53 cm long and 25 cm high. Plastic trays, lined with absorbent paper, were placed below each cage to collect animal excreta. The paper was changed daily. The cages were changed for clean cages on one occasion during the study.
- Diet: standard quality controlled laboratory rat food (SDS Laboratory Animal Diet No. 1 Special Diets Services, Witham, Essex), ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.5 - 20
- Humidity (%): 44 ± 18
- Air changes: separate ventilated cabinet
- Photoperiod: 12 hours light (0730 - 1930 h) and 12 hours dark per 24 hours

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Each exposure system employed consisted of a whole body inhalation chamber, chamber monitoring trolley and all glass vaporiser to produce an aerosol from the liquid test material. Accessories included an elutriator, air supply and extract lines which attached to the top and bottom of the chamber respectively.
- Source and rate of air: The air supply was provided by compressor. The air was filtered to remove any residual particulate and was dried.
- System of generating vapors: An all glass vaporiser was used to generate the vapour. The vaporiser was housed in a water bath at approximately 60°C. The liquid to be administered was supplied to the vaporiser from a polypropylene syringe (Groups 2 and 3) or from two glass syringes (Group 4) driven at a controlled rate by an infusion pump (Precidor type 5003). The generation apparatus was positioned under a heating lamp used to maintain the ambient temperature greater than 23 °C, to prevent solidification ofthe test substance. The vapour was carried to a glass elutriator attached to the inlet at the top of the chamber by flexible tubing.
- Temperature, humidity, pressure in air chamber: 21.5-23.2°C, relative humidity: 27-44%, pressure: 1 and 10 mm water below ambient
- Air flow rate: 150 Iitres/minute
- Treatment of exhaust air: At the end of the 6-hour exposure period the air supply to the vaporiser was disconnected and one of the chamber sampling ports opened to the atmosphere. The system was allowed to clear for approximately 20 minutes before the rats were returned to their holding cages. The chambers were washed with hot water.

TEST ATMOSPHERE
- Brief description of analytical method used: The samples of chamber atmosphere were injected directly into a gas chromatograph calibrated using vapour standards prepared in gas bags.
- Samples taken from breathing zone: yes, Chamber atmosphere was sampled in sequence from each of the four exposure chambers (Groups 4 - 1 sampled sequentially) from the right upper middle sampling port using gas tight polypropylene syringes (20mL) at approximately hourly intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Characterisation of the exposure chamber:
Preliminary trials were performed at the high dose level to investigate the spatial variation in vapour concentration within the exposure chamber (Note: trials were previously performed at the low and intermediate dose levels with satisfactory results - see Huntingdon Life Sciences report number ZCE 027/974090). From left to right, back to front and top to bottom there was no significant difference in the chamber vapour concentration. Accordingly, the vapour generation system was considered satisfactory for use in animal exposures.
- Chamber concentration of test item:
The study mean chamber concentrations of test item were close to target for each group. The coefficients of variation of the daily mean values were 5% or lower demonstrating good control of the exposure system.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

6 hours each day, for Days 6 to 19 of presumed pregnancy.

Duration of test:

20 day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (nominal)

Remarks:

Group 1 (Air control)

Doses / concentrations 2

Dose / conc.:

350 ppm (nominal)

Remarks:

Group 2 (Low dose)

Doses / concentrations 3

Dose / conc.:

1 000 ppm (nominal)

Remarks:

Group 3 (Inter dose)

Doses / concentrations 4

Dose / conc.:

3 500 ppm (nominal)

Remarks:

Group 4 (High dose)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: taking account of all available data, including the results of a preliminary study for the effect on pregnancy of the rat (inhalation exposure) performed with test item (Huntingdon Life Sciences report number: ZCE 027/974090).
- Animal assignment: Individual bodyweights were processed using a computer program which selected 40 or 20 rats for allocation to 4 groups, such that the group mean bodyweights were approximately equalized. Adjustments were made as necessary to ensure an acceptable distribution of the males to which the females were mated.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: checked in the morning and again at the end on the normal
working day
- Cage side observations: Signs of ill-health, together with any behavioural change or response to treatment, were recorded.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 of presumed pregnancy, and on Days 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 of presumed pregnancy.

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

WATER CONSUMPTION: Yes
- The amount of water was caIculated as the difference, over consecutive days, in the weight of the water bottle and contents given to each cage of rats.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: maternal organs

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number and distribution of live young: Yes
- Number and distribution of embryofetal deaths: Yes
- lndividual fetal weight (from which the liver weight was calculated): Yes
- Fetal abnormalities: Yes
- Uteri or individual uterine horns without visible implantations were examined for evidence of implantation using a modified Salewski technique.

Fetal examinations:

Live young were examined externally and weighed. Fetuses were uniquely identified to allow correlation of initial with subsequent findings. Half the fetuses in each litter were preserved in Bouin's solution for subsequent free-hand sectioning to discover visceral abnormalities by the Wilson technique; the remainder were fixed in 74 OP industrial methylated spirit for subsequent macroscopic examination, evisceration, clearing and alizarin staining by a modified Dawson technique for skeletal examination.
AII fetuses were sexed by gonadal inspection following preservation.

Statistics:

- Adult data:
Statistical tests employing analysis of variance followed by an intergroup comparison with control.
Dependent on the heterogeneity of variance between treatment groups, parametric tests [analysis of variance followed by Williams’ (Williams, 1971/2) or ‘t’ tests or non-parametric tests (Kruskal-Wallis test (Hollander & Wolfe, 1973)] were used to analyse these data, as appropriate.
- Litter data:
Litter data values were analysed by the Kruskal-Wallis test (Hollander & Wolfe, 1973). Intergroup comparisons were made either by the non-parametric equivalent of the ‘t’ test together with the Jonckheere test for an ordered series of treatments (Hollander & Wolfe, 1973) or Shirley's test (Shirley, 1977).
Where 75% or more of the values for a given variable were the same, Fisher's Exact test (Fisher, 1950) and Mantel’s test (Mantel, 1963) were used.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During most exposures Group 4 (High dose) rats were less responsive to tapping on the chamber door than control rats, showed an unsteady gait in response to tapping on the chamber door and had eyes half closed and a hunched posture. On one occasion animals at the front of the chamber were noted to be salivating. The Group 3 (Inter dose) rats were less responsive to tapping on the chamber door than controls and had their eyes half closed during most exposures. Hunched posture and an unsteady gait in response to tapping on the chamber door were each seen on a single occasion for Group 3 rats. The signs tended to occur earlier in the exposure for Group 4 rats compared with Group 3 and, once observed, were present until the end of the exposure for both groups.
One rat from Group 4 (High dose) was noted to have a growth on the dorsal surface of the shoulder region. This was considered spontaneous in origin and unrelated to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was one unscheduled death during the study. Death was not attributable to the administration of test item. Rat 95 of Group 4 (High dose) was killed for humane reasons on Day 16 of presumed pregnancy after exhibiting swelling of the left hindfoot from toe to hock.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Between Days 6 and 8 of pregnancy, bodyweight gain for Groups 3 (Intermediate dose) and 4 (High dose) was statistically significantly lower than control. This was sustained between Days 8 and 10 of pregnancy for Group 4. Between Days 10 and 20 of pregnancy there were no statistically significant differences in bodyweight gain.
No treatment-related effects on the pattern of bodyweight gain were observed for Group 2 (Low dose).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

With the exception of Days 16 to 19, the amount of food eaten by Group 4 (High dose) rats over each two-day period from Day 6 of pregnancy was statistically significantly lower than control. Over Days 6 to 19 of pregnancy, the cumulative amount of food consumed by Group 4 (High dose) rats was slightly, but statistically significantly lower than control.
No treatment-related effects on food consumption were observed for Groups 2 (Low dose) and 3 (Intermediate dose).

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Over Days 17 to 19 of presumed pregnancy the amount of water consumed by Groups 3 (Intermediate dose) and 4 (High dose) was higher than control. While the differences were probably treatment-related they did not attain statistical significance and consequently were considered to be of no toxicological importance.
No treatment-related effects on water consumption were observed for Group 2 (Low dose).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The type, incidence and distribution of macroscopic abnormalities noted in all treated groups at post mortem examination did not indicate any relationship to treatment.

Maternal developmental toxicity

Pre- and post-implantation loss:

effects observed, non-treatment-related

Total litter losses by resorption:

no effects observed

Dead fetuses:

effects observed, non-treatment-related

Changes in number of pregnant:

effects observed, non-treatment-related

Details on maternal toxic effects:

A total of 21, 21, 23 and 22 dams had live young at Day 20 in Groups 1 to 4 respectively. For the Group 4 (High dose), two rats were non-pregnant and one rat was killed early.
There were no instances of total litter loss in any group.
Gravid uterine for Group 4 (High dose) were statistically significantly lower than control.
There were no treatment-related changes for pre-implantation losses and number of corpora lutea. The general incidence of intra-uterine deaths was as expected for this strain in this laboratory and the observed inter-group differences were considered incidental.

Effect levels (maternal animals)

Dose descriptor:

NOEC

Effect level:

350 ppm (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Reduction in number of live offspring:

effects observed, non-treatment-related

Changes in sex ratio:

effects observed, non-treatment-related

Changes in litter size and weights:

effects observed, treatment-related

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The pattern of incidence of major malformations in this study did not índícate a relationship to treatment.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

There was evidence of some disturbance/delay in the process of skeletal ossification in Group 4 (High dose), as demonstrated by the incidence of incomplete ossification of some vertebral and sternal elements. Also in this group there was an increased incidence of certain minor abnormalities of the skeleton: small unossified fissure-shaped areas in the interparietal bone of the skull, and truncation of the anterior angle of the blade of the scapula; these changes did not appear together within the same fetus in any of the study groups, although in the majority of High dose group cases they did occur together within the same 4 litters. These minor abnormalities did not show any association with particularly low fetal weight, the weights of the affected fetuses within any of the groups being, on average, equivalent to the relevant group mean fetal weights. The occurrence of the scapula abnormality in Group 3 (Intermediate dose), at an incidence that was marginally in excess of that seen in the Control and Low dose groups, was considered to be of a doubtful biological significance, particularly in view ofthe absence of other fetal disturbances in the group.

Details on embryotoxic / teratogenic effects:

Litter and fetal weights for Group 4 (High dose) were statistically significantly lower than control, group mean fetal weight being reduced by about 14%.
There were no treatment-related changes for sex ratio, in utero deaths or live young.

Effect levels (fetuses)

Dose descriptor:

NOEC

Effect level:

1 000 ppm (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

skeletal malformations

Fetal abnormalities

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: sternum

skeletal: vertebra

Description (incidence and severity):

There was an increased incidence of incomplete ossification of vertebral and sternal elements in Group 4 (High dose).

Overall developmental toxicity

Developmental effects observed:

yes

Lowest effective dose / conc.:

3 500 ppm (nominal)

Treatment related:

yes

Relation to maternal toxicity:

not specified

Applicant's summary and conclusion

Conclusions:

It is concluded that administration of test item to the pregnant rat for 6 hours a day between Days 6 and 19 post coitum produced maternal bodyweight effects at 1000 and 3500 ppm and an associated effect on food consumption at 3500 ppm. With respect to the conceptus, there was evidence of some minor embryo-fetal toxicity at 3500 ppm, as indicated by the reduced fetal bodyweight with concomitant slight delay in skeletal ossification, and also by other minor disturbances in the skeleton, affecting the interparietal bone of the skull and the blade of the scapula. There was no definite evidence of an effect on embryo-fetal development at 1000 ppm (Inter dose) or less.

Executive summary:

The objective of this study was to assess the effect of test item upon the pregnant rat and the in utero development of the concepti based on OECD 414. Three groups of rats (each of 25 time-mated females) of the Crl: CD BR VAF/Plus strain, were exposed by whole body to a vapour of test item, 6 hours a day on Days 6 to 19 post coitum inclusive. On Day 20 post coitum,all females were sacrificed, subjected topost mortemexamination, litter values were determined and fetuses examined for visceral and skeletal changes. A fourth group acting as control was exposed to air only.  

One animal from Group 4 (High dose) was killed for humane reasons on Day 16 of presumed pregnancy. Post mortem examination revealed swelling of the left hindfoot which was not considered to be attributable to exposure to test item.

An effect of test item on bodyweight gain was evident between Days 6 and 8 of pregnancy for Groups 3 (Intermediate dose) and 4 (High dose) and this was sustained between Days 8 and 10 of pregnancy for Group 4. There was no effect on bodyweight gain between Days 10 and 20 of pregnancy.

The amount of food eaten by Group 4 (High dose) rats was slightly lower than control for most of the period of exposures except towards the end of the pregnancy.

There was an increased incidence of incomplete ossification of vertebral and sternal elements in Group 4 (High dose). Also in this group there was an increased incidence of certain minor abnormalities of the skeleton and these changes mostly occurred together within the same 4 litters. These minor abnormalities did not show an association with particularly low fetal weight, affected fetuses being, on average, equivalent to the group mean fetal weights.

It is concluded that administration of test item to the pregnant rat for 6 hours a day between Days 6 and 19 post coitum produced maternal bodyweight effects at 1000 and 3500 ppm and an associated effect on food consumption at 3500 ppm. With respect to the conceptus, there was evidence of some minor embryo-fetal toxicity at 3500 ppm, as indicated by the reduced fetal bodyweight with concomitant slight delay in skeletal ossification, and also by other minor disturbances in the skeleton, affecting the interparietal bone of the skull and the blade of the scapula. There was no definite evidence of an effect on embryo-fetal development at 1000 ppm (Inter dose) or less.