Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 5 August, 2004 to 23 June, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: It was conducted according to OECD test guideline,and with GLP compliance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Fetus 4521-13 was eviscerated prior to recording the sex, which could not then be determined. Fetus 4507-4 was switched from visceral examination to skeletal examination. Supplier did not provide a Gestation Day 0 weight list for 24 animals.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: gas
Details on test material:
Supplier: Honeywell P.O. BOX 2830 Baton Rouge, LA 70821
Lot number: BR4141;
Purity 99.95%
MW: 166.47
Expiration date: 30 June 2005
Storage conditions: room temperature

Test animals

Species:
rat
Strain:
other: Sprague-Dawley CD
Details on test animals and environmental conditions:
FACILITIES MANAGEMENT/ANIMAL HUSBANDRY
Currently acceptable practices of good animal husbandry were followed e.g., “Guide for the Care and Use of Laboratory Animals”, National Academy Press, 1996. Huntingdon Life Sciences, East Millstone, New Jersey is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).

HOUSING
Animals were housed individually in suspended stainless steel wire mesh cages.
FEED
Certified Rodent Diet, No. 5002; (Meal) (PMI Feeds, Inc., St. Louis, MO) ad libitum.

WATER
Facility water was supplied by Elizabethtown Water Company, (Westfield, NJ) and provided ad libitum to individual animal cages through an automated watering system.

ENVIRONMENTAL CONDITIONS

Light/Dark Cycle
A twelve-hour light/dark cycle controlled via an automatic timer was provided.

Temperature
Temperature was monitored in accordance with Testing Facility SOPs and maintained within the specified range to the maximum extent possible.
Desired Range: 18 to 26°C
Actual Range: 20.3 to 22.8°C
Daily Average Range: 21.2 to 21.9°C

Relative Humidity
Relative humidity was monitored in accordance with Testing Facility SOPs and maintained within the specified range to the maximum extent possible.Recordings: outside the specified range were not considered to have affected the integrity of the study.
Desired Range: 30 to 70%
Actual Range: 48.80 to 89.31%
Daily Average Range: 49.36 to 58.05%

Air Changes
Animal quarters had 10-15 air changes per hour. The actual number of air changes per hour in each animal room is recorded at least twice each year and the Testing Facility retains these records.

Additional details on animals:

NUMBER OF ANIMALS
Ordered: 104 time-mated rats.
Placed on study: 100 time-mated rats.
AGE AT RECEIPT
Time-mated female rats received on GD 0, 1 or 2 were between 75-90 days in age.
WEIGHT AT INITIATION OF EXPOSURES
Females weighed between 231-300 grams on Gestation Day 6.

Animals were acclimated for a minimum of 4 days from time of receipt until initiation of exposures on Gestation Day 6.


Administration / exposure

Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
The whole-body exposure chambers each had a volume of approximately 1000 Liters. Each chamber was operated at a minimum flow rate of 200 Liters per minute. The final airflow was set to provide at least one air change (calculated by dividing the chamber volume by the airflow rate) in 5.0 minutes (12 air changes/hour) and a T99 equilibrium time of at most 23 minutes. This chamber size and airflow rates were considered adequate to maintain the animal loading factor below 5% and the oxygen level at 19% or higher. At the end of the exposure, all animals remained in chamber for a minimum of 23 minutes. During this time, the chamber was operated at the same flow rate as used during the exposure using clean air only. Recordings of airflow rate and static pressure were made at the initiation of the exposures and every half-hour during the exposure. The chamber atmospheres were exhausted through the in-house filtering system, which consisted of a coarse filter, a HEPA filter, and an activated charcoal bed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Determination of the exposure levels were made using a MIRAN® Ambient Air analyzer equipped with a strip chart recorder. The test atmosphere was drawn from the normal sampling portal through the MIRAN® and measurements were recorded at least 4 times during each exposure. The exposure levels were determined by comparison of the measured absorbance to a calibrated response curve constructed using the same instrument settings.
Details on mating procedure:
Time-mated female rats received from provider on GD 0, 1 or 2 were between 75-90 days in age. Thus, the mating procedure did not take place in this test.
Duration of treatment / exposure:
days 6-19 of gestation
Frequency of treatment:
6 hours/day, 7 days/week
Duration of test:
during gestation days 6-19
No. of animals per sex per dose:
Each group contained 25 time-mated female rats.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Based on the existing information of subacute and subchronic studies for CTFE (Gad et al., 1988; section 7.5.2), a high exposure level of 180 ppm was selected for this study to provide slight maternal toxicity, while lower levels of 90 and 30 ppm were selected to provide a range of possible effects, including a NOEC or NOAEC.
- Rationale for animal assignment: random.

ANIMAL ASSIGNMENT
Animals were placed into study groups after receipt using a computerized randomization program. This ranked GD 4 body weights into blocks and randomly assigned each animal within each block into one of the study groups. Disposition records of all animals not utilized in the study are maintained in the study file.
ANIMAL IDENTIFICATION
Each animal was assigned a temporary identification number upon receipt. After selection for study, each animal was ear-tagged with a number assigned by the Testing Facility. This number plus the study number comprised a unique identification for each animal. Each study animal’s cage was also allocated a cage card, which was color-coded for exposure level identification and contained the study number and animal number.

Examinations

Maternal examinations:
Animals were observed in their cages at least twice daily for mortality, morbidity and signs of severe toxicity.

In-Chamber: All animals were observed as a group at least once during each exposure.
Out-of-Chamber: Animals on study were examined daily from receipt through to terminal euthanasia on GD 20. Examinations included observations of general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia as well as evaluations of respiration. During the treatment period these evaluations were performed after exposures.

Body weights were recorded on GD 4, 6, 10, 13, 16, 19 and 20. Weight gain was also calculated for intervals as found appropriate, including GD 6-20.
Feed consumption was recorded and reported for intervals as found appropriate, eg GD 4-6, 6-10, 10-13, 13-16, 16-20 and 6-20.
Ovaries and uterine content:
For all Gestation Day 20 animals, the intact uteri (ovaries attached) were removed from the abdominal cavity and weighed. Corpora lutea were counted and the number per ovary recorded. The number and location of the following were recorded for each uterine horn:
1. live fetuses;
2. dead fetuses (no significant degeneration);
3. late embryo-fetal deaths (recognizable dead fetus undergoing degeneration, regardless of size);
4. early embryonic deaths (evidence of implantation but no recognizable fetus)
Females that did not deliver pups were sacrificed and their uteri examined for implantation sites and resorptions, including confirmation of any apparent non-pregnant status by means of staining with ammonium sulfide (method modified from Salewski, 1964) to detect nonvisible implantation sites. The maternal carcass and uterus were then discarded.
Fetal examinations:
All live fetuses were weighed and individually identified. Each (live and dead) fetus was given a macroscopic external examination for defects, including observation of the palate. Any dead fetuses were also weighed.

Visceral evaluations:
Approximately one-half of the fetuses in each litter (nominally, alternating fetuses within the litter were randomly assigned using a computerized randomization program - Toxicology Analysis System Customized, version 1.4) and were placed in suitable aldehyde (such as Modified Davidson's fixative) for preservation and decalcification. These fetuses were subjected to soft tissue examination by gross dissection of the torso and a razor blade sectioning technique for the head. All malformations and variations were recorded. During the dissection process, the sex of each fetus was confirmed by internal inspection of the gonads. Following complete dissection of the fetuses, all carcasses and sections were preserved in 10% neutral buffered formalin.

Skeletal evaluations:
Approximately one-half of the fetuses in each litter (nominally, alternating fetuses within the litter were randomly assigned using a computerized randomization program - Toxicology Analysis System Customized, version 1.4) had the sex internally evaluated and were then eviscerated and processed for staining of the skeleton using Alizarin Red S. Subsequently, these fetuses were evaluated for skeletal malformations and ossification variations. These specimens were then stored in 100% glycerin with a mold inhibitor.
Statistics:
The following parameters were analyzed statistically, where it was considered necessary in support of interpretation. Statistical significance for differences from control was recognized at the 5% or 1%, two-sided levels.
a) Continuous data
Evaluation of equality of group means was made by the appropriate statistical method, followed by a multiple comparison procedure, if needed. The parametric method was the standard one-way analysis of variance (ANOVA) using F ratio to assess significance (Dunlap and Duffy, 1975). If the ANOVA was significant, Dunnett's test (Dunlap, et.al., 1981) was used to determine which data, if any, differed from the control. The nonparametric method was the Kruskal-Wallis test (Siegel, S., 1956) and if differences were indicated, the pairwise comparison test (Siegel, S., 1956) was used to determine which means differed from control.
b) Incidence data
A Fisher's Exact Test with Bonferroni correction (Siegel, S., 1956) was performed to identify differences between the groups.
Incidence of litters containing fetuses with malformations (external, soft tissue and skeletal - total affected and by finding)
Indices:
No data
Historical control data:
no data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: Decreased body weight gain and feed consumption at 90 and 180 ppm.

Details on maternal toxic effects:
There was no effect of treatment on survival, as all animals survived until the termination of the study. Pregnancy rates varied from 88-96% with no treatment effect seen. During the exposure periods, all animals were unremarkable except the 180 ppm exposed dams showed some eye closure, piloerection and lethargy during exposure day 7 prior to this exposure level's early termination. The 90 ppm exposed animals showed a marked increase in red nasal discharge after removal from the exposure chambers, especially during the period of gestation days 13-20. There were statistically significant effects on body weights during gestation days 6-10 (corresponding with the initial 1-4 exposures) for the 90 ppm group (15% less weight gain than control) and 180 ppm group (7% weight loss compared to control).
The 180 ppm exposed dams showed some decreased feed consumption and consequent decreased fecal volume.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEC
Effect level:
31.1 ppm (analytical)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Effect level:
30 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Effect level:
89.2 ppm (analytical)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEC
Effect level:
90 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
Significantly reduced (13%) fetal body weight in the 180 ppm exposed animals, compared to the Air Control animals. There was also slight reduction in the state of ossification of the fetal skeleton at the 180 ppm exposure level, but for the sporadic incidences of other fetal variants and abnormalities there was no indication of association with test substance exposure.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEC
Effect level:
180 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Dose descriptor:
NOAEC
Effect level:
187 ppm (analytical)
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 at 180 ppm resulted in significant body weight loss in the dams. As a consequence, exposure to this group was terminated prior to the final scheduled exposure. However the rats were evaluated at the end of the gestation period along with the rats from the other groups. There was no evidence for developmental abnormalities at any level. Decreased body weight gain and food consumption was reported for the dams exposed to 90 and 180 ppm of CTFE. There was also slight reduction in the state of ossification of the fetal skeleton at the 180 ppm exposure level, but for the sporadic incidences of other fetal variants and abnormalities there was no indication of association with test substance exposure.

There were no test substance related effects on pregnancy outcome, in terms of corpora lutea numbers, pre- or post- implantation loss, or litter size and gravid uterine weights. The slightly increased numbers of early embryonic deaths that occurred in the exposed groups, as compared with Air Control, were the result of a small minority of animals in those groups with more than the usual number of eaily deaths:

 

Air control

30 ppm

90 ppm

180 ppm

Animals with

1 early death

5

4

7

6

Animals with

>learly

deaths

0

4

5

2

It is not uncommon for rats of this strain to have at least 1 early embryonic death in each pregnancy, often affecting over half of the litters in the group. In view of this, and the lack of an exposure concentration relationship, the incidences in this study were considered to have been fortuitous.

Experimental data are reported in the field "Attached background material".

Applicant's summary and conclusion

Conclusions:
There was no evidence for developmental abnormalities at any level.
Executive summary:

A prenatal toxicity test was conducted according to OECD test guideline 414, which was compliant with GLP. In the test, groups of 25 time-mated female rats were exposed once daily for 6 hours/day, 7 days/week during gestation days 6-19 and euthanized and necropsied on gestation day 20. The following parameters were evaluated: viability, clinical observations, body weights and feed consumption. Exposure levels were determined using an IR sampling procedure 4 times per chamber per day. Particle size distribution measurements were also made once per chamber per week. At caesarean section, the animals were subjected to a macroscopic postmortem evaluation and corpora lutea/implantation data were recorded. The fetuses were examined for externally visible abnormalities, weighed and then examined for soft-tissue abnormalities using a microdissection procedure. Their skeletons were then stained with Alizarin Red S and examined for skeletal abnormalities and ossification state; there was no treatment effect on survival.

The mean (± standard deviation) analytical (IR) concentrations for the control and the respective exposure groups were as follows: 0±0, 31.1±2.3, 89.2±8.1 and 187±21 ppm. Exposure at 180 ppm resulted in significant body weight loss in the dams. As a consequence, exposure to this group was terminated prior to the final scheduled exposure. However the rats were evaluated at the end of the gestation period along with the rats from the other groups. There was no evidence for developmental abnormalities at any level. Decreased body weight gain and food consumption was reported for the dams exposed to 90 and 180 ppm of CTFE. There was also slight reduction in the state of ossification of the fetal skeleton at the 180 ppm exposure level, but for the sporadic incidences of other fetal variants and abnormalities there was no indication of association with test substance exposure.

Based on above results, exposure at 30 ppm was considered as the no-observed-adverse-effect-concentration ((NOAEC) for maternal toxicity, exposure at 90 ppm as NOAEC for developmental toxicity, and 180 ppm as NOAEC for teratogenicity.