Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 April 1977 and 1 June 1977
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
other company data
Title:
Unnamed
Year:
1977
Report Date:
1977

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
not specified
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
EDB was obtained from Aldrich Chemical Co., Milwaukee, Wis., with a purity of 99% and no further analysis was performed.

Test animals

Species:
other: Rats and Mice
Strain:
other: Rats: Charles River CD, Mice: CD-1
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River CD rats (Charles River Breeding Laboratories, North Wilmington, Massachusetts)
- Age at study initiation: not reported
- Weight at study initiation: not reported.
- Fasting period before study: not reported,
- Housing: Rochester type stainless steel chambers with a volume of about 3.5 m3 were used in this study.
- Diet/Water: Animals were given free access to powdered rodent chow (Wayne Lab-Blox, Allied Mills, Inc., Chicago, Illinois), and tap water except where indicated in the experimental protocol. During the treatment period, feed was changed daily in order to prevent possible accumulation of EDB in the feed. The control group was subdivided into animals given free access fo feed and animals whose feed was restricted.
- Acclimation period: At least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The quarters were maintained at 72°F
- Humidity (%): relative humidity of 50 ± 10%
- Air changes (per hr): Clean air at a flow rate of 10 to 12 changes per hr entered at the top of the chamber.
- Photoperiod (hrs dark / hrs light): 7:00 AM to 7:00 PM photoperiod.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
Rochester type stainless steel chambers with a volume of about 3.5 m3 were used in this study. Clean air at a flow rate of 10 to 12 changes per hr entered at the top of the chamber. EDB vapor was generated by bubbling nitrogen into a glass vessel which was maintained at 30°C. EDB entered the air stream upstream from the chamber. Mixing was initiated in a plenum at the top of the chamber and completed by two squirrel cage fans and a diffusion plate.

The EDB concentration in the chamber was monitored using gas chromatography and a flame ionization detector. EDB was resolved using a stainless steel column packed with 5% didecyl phthalate on 80/100 chromosorb and nitrogen (80 ml/min) as the carrier gas. The injection, column and detector temperatures were 160°C, 145°C and 170°C, respectively. Standards were prepared by serial dilutions of an EDB stock solution prepared in carbon tetrachloride.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The EDB concentration in the chamber was monitored using gas chromatography and a flame ionization detector.
Details on mating procedure:
Female rats and mice were exposed overnight to proven male breeders. Successful mating was identified the next morning (day 0 of gestation) by the presence of sperm in vaginal smears
Duration of treatment / exposure:
Animals were housed in the inhalation chambers for 10 days starting on Day 6 of gestation.
Frequency of treatment:
23 hours a day
Duration of test:
Rats were sacrificed on gestational day 20.
Doses / concentrations
Remarks:
Doses / Concentrations:
20, 38 & 80 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
Between 15 & 17 animals per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
Mated animals were divided into control and treated groups. The control group was subdivided into animals given free access fo feed and animals whose feed was restricted. The EDB treated group was subdivided into animals exposed to 20, 38, or 80 ppm of EDB.

Examinations

Maternal examinations:
Maternal animals were observed for adverse effects on welfare and reproduction. Bodyweight and feed consumption was also monitored.
Ovaries and uterine content:
A laparotomy was performed and the uterine horns were exposed.
Fetal examinations:
The umbilical cord was clamped and severed distally in order to prevent blood loss. Fetuses were removed, weighed and examined for external anomalies. One half of the fetuses from each litter was fixed in Bouin's solution and examined for soft-tissue anomalies by a free-hand slicing method. The remaining fetuses were fixed in 70% alcohol, eviscerated, stored in 1% KOH and stained with alizarin red. After differential decolorization, the skeletons were examined for anomalies.
Statistics:
Quantitative data, reported as the mean ± standard error, were initially analyzed by Bartlett's test for homogeneity. The test of significance for homogeneous data was Dunnett's procedure. In contrast, heterogeneous data were analyzed by the two-sample rank test. Enumeration data were analyzed with the Fisher's exact probability test. For all tests the 0.05 level of significance was chosen except where indicated. The litter was considered to be the unit of observation. All statistical tests, therefore, were based on the litter as the experimental unit.
Indices:
not reported.
Historical control data:
not applicable.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Rats:
Adverse effects on maternal welfare and reproduction were observed in rats exposed to EDB (Table 1). Deaths occurred only at the high concentration. A weight loss was evident in tats exposed to 38 and 80 ppm of EDB. In addition, the weight gain at the end of exposure was reduced at the high concentration. Feed consumption was reduced in rats exposed to all concentrations of EDB and remained depressed in the high concentration group when the exposure was terminated. Dams exposed to 80 ppm of EDB had a reduced number of implants and evidence of embryotoxicity, as measured by increased resorptions. The body weight of fetuses from dams exposed to 38 ppm was reduced. In the feed restricted group, feed consumption and weight gain were reduced during organogenesis. When these rats were given free access to feed a compensatory weight gain occurred. Although fetal body weights were reduced, there was no evidence of embryolethality.

Mice:
In mice, death occurred in groups exposed to 38 and 80 ppm of EDB as well as the group with restricted feed consumption (Table 2). In the last group deaths were due to canibalism. The weight change was reduced in the 20 and 38 ppm EDB exposed groups and the feed restricted group. The weight change after the exposures were terminated was normal in all groups except the one previously exposed to 38 ppm of EDB. In the group exposed to 20 ppm of EDB the percent of late resorptions was increased and fetal body weights were reduced. In the group exposed to 38 ppm of EDB, the percent of viable fetuses was reduced, the incidence of resorptions was increased, fetal body weights were reduced, and the percent of male fetuses was increased. In the feed restricted group the percent of viable fetuses was reduced and the incidence of resorptions was increased. Although only one dam produced viable fetuses, these fetuses had a reduced body weight and a reduced percent of males.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
LOAEC
Effect level:
20 ppm
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEC
Effect level:
20 ppm
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Rats:
Hematoma were the most common external anomaly that occurred in rats. They occurred in a variety of regions and were present in all of the groups. The incidence (fetuses affected/fetuses inspected) of umbilical hernia and clubbed feet in the group exposed to 38 ppm of EDB were 1/184, and 2/184, respectively. The soft tissue anomalies observed in rats are presented in Table 3. None of these anomalies was present in any of the treated groups at an incidence that reached a level of statis¬tical significance.

The skeletal anomalies observed in rats are reported in Table 4. A reduced percent of fetuses with normally ossified centri occurred in the group exposed to 20, but not 38 ppm of EDB. In contrast this parameter was increased in the feed restricted group. In general, EDB exposure did not dramatically alter the incidence of skeletal anomalies.

Mice:
Few external anomalies were observed in mice. Exencephally occurred in three of 218 fetuses examined from the group exposed to 20 ppm of EDB. Hematomas occurred in a few of the fetuses from groups exposed to 0 and 20 ppm of EDB.

The soft tissue anomalies observed in mice are presented in Table 5. There was a high percent of runts among the fetuses from the group exposed to 38 ppm of EDB. This may be a reflection of the reduced fetal body weight which in turn may be a reflection of the reduced feed consumption and weight gain of the dams. None of the remaining anomalies listed occurred at statistically significant rate in either of the groups exposed to EDB.

The skeletal anomalies observed in mice are reported in Table 6. EDB exposure was associated with a statistically significant increase in some of these anomalies. Of these anomalies, incomplete ossification of supraoccipitals, unossified incus, and sternebrae anomalies (unossified and incompletely ossified) were previously reported in mice exposed to 31.6 ppm of EDB10/ (Appendix I Table 4). Unfortunately, there were no fetal mice in the present study to evaluate the effect of feed restriction on development. However, the results of the previous study suggested that the incidence of sternebrae anomalies was increased by restricting the availability of feed.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Rats inhaled EDB 23 hr a day for 10 days starting on Day 6 of gestation.

The values for the chamber concentration, which were determined at intervals during the exposure, were averaged to yield a time-weighted average concentration for that day. The average (range) of these values for each chamber during the 17-day period required to expose all of the animals was 20 (15-22), 38 (32-42), and 80 (7 l-84) ppm of EDB.

Applicant's summary and conclusion

Conclusions:
EDB produced adverse effects on maternal welfare, as measured either by weight change or feed consumption, in rats at all of the doses tested.
EDB was judged to have little primary effect on development in this study.
Executive summary:

Pregnant rats inhaled ethylene dibromide (EDB) at concentrations of 20, 38, and 80 ppm for 23 hr a day. The exposures started on Day 6 of gestation and lasted for a total of 10 days. Observations were made on maternal welfare and fetal development. The results of this study indicated that adverse effects on maternal welfare, as measured by weight gain, feed consumption, and survival, were

observed in rats; and that morphological changes were observed in fetuses from dams exposed to EDB. However, the latter occurred at concentrations that also affected maternal welfare. Consequently, EDB was judged to have little primary effect on development in this study.