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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15th January 1979 - 11th February 1980
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1980
Report Date:
1980

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Principles of method if other than guideline:
The exposure period was increased from to 95-96 days (13 weeks).
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Production grade ethylene dibromide supplied by The Dow Chemical Company, Magnolia, Arkansas was used for this study. Gas chromatographic analysis of the test material was made prior to study initiation. The test material was also analyzed twice during the study and after the final exposure by the Dow Analytical Lab. Analysis revealed the purity to be >99%.

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 9 weeks old
- Weight at study initiation: Males: 179-204g; Females: 121-135g
- Fasting period before study: Food (Purina Rat Chow) was withheld during the 6-hour exposure period.
- Housing:
The animals were housed 2/cage during non-exposure periods and 5 or 10/cage for females and males, respectively, during exposure periods.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Rats were acclimated to the conditions of this laboratory for at least 13 days prior to the initial exposure

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 29 °C
- Humidity (%): 40 - 51%
- Air changes (per hr): nda
- Photoperiod (hrs dark / hrs light): nda

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: Compressed air
Remarks on MMAD:
MMAD / GSD: Not reported
Details on inhalation exposure:
EDB was vaporized by metering the liquid at a calculated rate with a precision pump into a warmed vaporization flask (100°C). The vapors were swept
from the flask with compressed air into the main chamber airflow. The chambers were 1 m3 stainless steel and glass Rochester-type chambers.
The nominal concentration was calculated from the rate at which liquid EDB was dispensed and the total chamber airflow.
The chamber concentration of EDB was analyzed at least 3 times/day by gas chromatography using a flame ionization detector. A 6' x 1/8" 0D nickel column packed with 10% SP-1000 on 100/200 mesh Chromosorb W (Supelco, Inc.) was used for the analysis. The carrier (helium), hydrogen and air flows were 54, 20, and 300 ml/min, respectively. The column and detector temperatures were 140 and 200°C, respectively. The retention time for the EDB peak was approximately one minute after injection of the air sample.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The nominal concentration was calculated from the rate at which liquid EDB was dispensed and the total chamber airflow. The chamber concentration
of EDB was analyzed at least 3 times/day by gas chromatography using a flame ionization detector. A 6' x 1/8" 00 nickel column packed with 10% SP-1000 on 100/200 mesh Chromosorb W (Supelco, Inc.) was used for the analysis.
Duration of treatment / exposure:
Exposures were 6 hours/day, 5 days/week for 13 weeks for a total of 67-68 exposures in 95-96 days. Scheduled sacrifices occured after 1, 6, and 13 weeks of exposure. Additional rats were held for a recovery period of 88-89 days and subsequently necropsied.
Frequency of treatment:
Exposures were 6 hours/day, 5 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
3, 10 and 40 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
40 male and 20 female per dose level.
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale:
The CDF strain was chosen because of its current use in chronic toxicity/carcinogenicity studies in this laboratory and many other laboratories.

- Rationale for animal assignment (if not random):
The animals were randomly assigned to the four groups using numbers generated by the program GRAND.CLIST (Computation Laboratory, The Dow Chemical Company).

- Rationale for selecting satellite groups:
Serial sacrifices of 10 male rats/group were conducted after 1 (5 exposure days), 6 (29 exposure days) and 13 weeks (67 exposure days) and 10 female rats/group after 13 weeks (68 exposure days); the remaining 10 rats/sex/group were held and sacrificed after an 88-89 day post-exposure period for the purpose of assessing reversibility of any lesions that may be associated with exposure to EDB for 13 weeks.

- Post-exposure recovery period in satellite groups:
88-89 day post-exposure period
Positive control:
not applicable.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed at the end of each exposure period and signs of toxicity noted and recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Particular attention was paid to the eyes and nose for clinical indications of irritation.

BODY WEIGHT: Yes
- Time schedule for examinations: Rats were weighed twice prior to the start of the study, twice weekly for the first two weeks of the study and once weekly thereafter. Male rats exposed to the various concentrations of EDB for 5 days were initially exposed to EDB 6 days after the other groups were initially exposed. Consequently, their body weights were statistically analyzed separately from the other groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Basic hematological determinations were conducted on 7 male rats/exposure level prior to their necropsy after 6 weeks and also on 7 rats/sex/exposure level prior to their necropsy after 13 weeks of exposure.
- Parameters examined: Hematological parameters included a total erythrocyte count (ROC), total (WBC) and differential leukocyte counts, hemoglobin concentrations (HGB), and packed cell volume (PCV).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Clinical chemistry determinations was performed on all 10 animals/sex (if applicable)/exposure level at the time of necropsy for the serial sacrifices after 1, 6 and 13 weeks.
- Parameters examined: Clinical chemistry measurements included blood urea nitrogen (BUN), serum glutamic pyruvate transaminase (SGPT), serum glutamic oxalacetic transaminase (SCOT), serum alkaline phosphatase (AP), glucose, and bilirubin. Serum bromide levels of rats exposed to EDB for 6 weeks were measured by neutron activation.

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalysis determinations were conducted on 7 male rats/exposure level prior to their necropsy after 6 weeks and also on 7 rats/sex/exposure level prior to their necropsy after 13 weeks of exposure. Urinalysis on 7 rats/sex/exposure level was performed prior to the necropsy on animals in the recovery group.
- Parameters examined: Urinary paramters measured included specific gravity, pH, glucose, ketones, bilirubin, urobilinogen, occult blood and protein.



Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Gross examination of the eyes of all rats was performed by a microscope slide technique at necropsy with observations recorded as part of the gross pathologic examination. At the time of each sacrifice, the eyes from 5 rats/sex (if applicable)/group were preserved in Zenker's solution. The eyes of the remaining rats were preserved in 10% formalin. Gross necropsies were performed on all rats, with special attention given to assessing the presence or absence of inflammation of the upper respiratory tract. Rats were fasted overnight prior to necropsy. They were anesthetized with methoxyflurane and decapitated after clamping the trachea. The lungs and trachea of all animals were removed as a unit and inflated with 10% formalin from a hand held syringe. The nasal passageways were perfused with formalin fixative. Fasting body weights and organ weights for liver, kidneys, brain, heart, thymus, and testes (males) were obtained from all rats at each necropsy.

HISTOPATHOLOGY: Yes
Representative specimens of the tissues indicated below were taken from all animals and fixed in phosphate-buffered 10% formalin. The target tissues (lungs, bronchi, trachea, nasal turbinates, liver, kidney, testes, ovaries, uterus and oviducts) were processed by conventional histological methods, stained with hematoxylin and eosin and examined by light microscopy from all 10 rats/sex/group of each of the serial sacrifices after 1, 6, 13 weeks and the recovery group. Transverse sections through the decalcified nasal cavity were made perpendicular to the plane of the hard palate and the plane of the nasal septum at or near the following levels:
- immediately caudal to the upper incisor teeth
- at the incisive papilla
- at the second palatal ridge
- at the first upper molars.

Upon examination of the nasal turbinates, special attention was given to assessing the presence or absence of discernible inflammatory, degenerative, necrotic, hyperplastic or metaplastic changes.

Tissue specimens examined:
esophagus, salivary glands, stomach, small intestine, large intestine, pancreas, liver, kidneys, urinary bladder, prostate, accessory sex glands,
epididymides, testes, ovaries, oviducts, cerebrum, brain, cerebellum, brain stem, pituitary gland, spinal cord, peripheral nerve, trachea, lungs (bronchi), nasal turbinates, sternum, spleen, thymus, lymph nodes (thoracic, mesenteric), heart, aorta, skeletal muscle, adrenal glands, thyroid gland, parathyroid gland, adipose tissue, skin, any gross lesion or mass, uterus.

Statistics:
Body weights, body weight gain, organ weights, urine specific gravity, hematology and clinical chemistry data were evaluated using an analysis of variance and Dunnett's test (Steel and Torrie, 1960). The level of significance was p<0.05.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
- Animal Observations:
Male rats exposed to 40 ppm EDB for 5 days exhibited eye and nasal irritation during the first expsure period. This was not
observed in other animals scheduled for longer exposure or at different concentrations of EDB. No other effect relatable to ethylene dibromide
exposure was observed. Two non-exposure related deaths were observed in female rats from the 3 and 10 ppm exposure groups during the 88 day recovery period.

- Body Weights:
A decrease in body weight was observed in male rats exposed to 40 ppm EDB during the 13-week exposure period. Male rats exposed to 10 ppm EDB showed a significant decrease for the first two weeks of the study and occasionally thereafter. A statistically significant decrease in body weight was observed in female rats inhaling 40 ppm EDB on day 4 of the study. However, these significant differences were not nearly so evident when body weight gains were statistically analyzed. The male rats inhaling 40 ppm ethylene dibromide still showed statistically decreased body weight gain during the 13-week exposure period but no change in body weight gain was observed at lower concentrations. The female rats inhaling 40 ppm EDB showed a decrease in body weight gain on day 4 and 14 of the study.

- Hematology:
The female rats exposed to 40 ppm EDB for 13 weeks had a statistical decrease in hematocrit and in hemoglobin that may have
been the result of exposure. No other measured hematolgical parameters revealed an effect attributable to exposure to EDB; the
statistical increase in hematocrit of males exposed to 3 ppm EDB for 13 weeks and the statistical decrease in total white blood cell counts
of males exposed to 3 or 10 ppm EDB for 6 weeks were considered to be of no toxicologic significance due to a lack of a dose response
and the expected variability in this parameter.

- Urinalysis:
No treatment-related effects on urinalysis parameters were observed in any male rats exposed to EDB. However, female
rats exposed to 40 ppm EDB for 13 weeks showed a statistically significant decrease in specific gravity of the urine which was interpreted
as treatment-related. After a recovery period of 88 days, the decrease in specific gravity was not observed.

- Clinical Chemistry:
As expected, serum bromide levels were significatly elevated in a dose-related manner above control values for all groups of
male rats exposed to EDB for 6 weeks. No other parameters, of those measured, exhibited a consistent effect due to exposure to
ethylene dibromide; the statistical increases in total bilirubin noted in rats exposed to 3 or 10 ppm EDB for 13 weeks and the statistical
decrease in SGPT values in rats exposed to 40 ppm EDB for 1 week were considered representative of the normal variation seen with these
parameters and of no toxicologic significance.

- Organ/Body Weight Ratios:
After one week of exposure, there were no statistically significant differences in organ weights at any exposure level, although
the absolute and relative liver weights were elevated slightly. The relative liver weights of male rats exposed to 40 ppm EDB were increased at each of
the 3 subsequent necropsy intervals; this was considered to be the result of the exposure to 40 ppm EDB and the accompanying decrease in body
weight of this group. Female rats exposed to 10 or 40 ppm EDB for 13 weeks had elevated absolute (40 ppm only) and relative liver weight
values. These liver weight changes observed in the female rats after exposure to 10 or 40 ppm EDB for 13 weeks were most likely due to
exposure; these changes were not observed at necropsy in rats after an 88 day recovery period. Kidney weights of male rats were increased after 6 weeks of exposure to 3 (relative basis only), 10 (relative basis only) and 40 ppm (absolute and relative basis) EDB. However, after 13 weeks of exposure, kidney weights were increased on a relative basis only in males exposed to 40 ppm EDB. Thus, the transient increase in relative body weights
noted after 6 but not 13 weeks of exposure to 3 or 10 ppm EDB was considered to be of questionable significance. Kidney weights of female rats were not statistically different from control values at any of the exposure levels, but there was a trend toward increased relative kidney weights in female rats exposed to 40 ppm EDB for 13 weeks.

The weights of the brain, heart, thymus and testes were not considered to be directly affected by exposure to 3, 10, or 40 ppm EDB. The
statistical increase in relative brain weights noted in males exposed to 40 ppm EDB for 6 or 13 weeks or 10 ppm EDB for 6 weeks were considered
secondary reflections of the lower body weights of these groups. The statistical increase in relative weight of the thymus of male rats exposed
to 10 ppm EDB for 13 weeks was considered an expression of the normal variability historically encountered in recording the weight of the
thymus. The increase in relative weights of testes noted in the recovery group subsequent to exposure to 3, l0, or 40 ppm EDB were considered
to be a secondary reflection of the lower body weight as compared to the control group sacrificed at that time. The same explanation applies to
the decreased absolute testicular weights in males exposed to 40 ppm EDB for 13 weeks.


- 6-day Interim Sacrifice:
There were no grossly visible lesions considered to be related to treatment. Effects attributable to EDB were seen only in the most anterior section of the nasal turbinates. All male rats exposed to 40 ppm EDB showed very slight to slight scattered to diffuse hyperplasia of the respiratory epithelium of the turbinates. Five of ten male rats of this group showed very slight focal individual epithelial cell necrosis of the respiratory epithelium. Nine of ten male rats exposed to 10 ppm EDB showed isolated to scattered hyperplasia of the re3piratory epithelium graded very slight fo slight in degree. One rat of this group showed focal individual epithelial cell necrosis of the respiratory epithelium, very slight in degree. Examination of sections of nasal turbinates of rats exposed to 3 ppm EDB revealed no hyperplasia or other lesions related to exposure.

Most of the male rats of the control group showed varying distribution of slight submucosal and epithelial inflammation of the respiratory epithelium vlith focal aggregates of inflammatory cells in the lumen of the nasal turbinates. In addition, the tracheal submucosa of all male
control rats showed a similar inflammatory response, accompanied by a hyperplastic response of the mucosal epithelium. This inflammation
occurs at a highly variable rate in this laboratory. Male rats exposed to 3, 10 or 40 ppm showed a substantial decrease in the inflammatory
reaction noted in both nasal turbinates and trachea in comparison to controls. This decrease in inflammation noted in the nasal turbinates
and trachea may or may not have been the result of exposure to EDB. All other histopathologic observations were considered to be spontaneous in
nature and typical of rats of this age and strain.

- 40-Day Interim Sacrifice:
There were no gross observations which were considered to be the result of exposure. EDB exposure-related effects were again limited to the most anterior section of the respiratory epitnelium of the nasal turbinates. All male rats exposed to 40 ppm EDB showed very slight to slight multifocal to diffuse hyperplasia and very slight to slight multifocal individual epithelial cell necrosis of the respiratory epithelium. All male rats exposed to 10 ppm EDB showed very slight to slight hyperplasia of the respiratory epithelium with an isolated to diffuse distribution. Nasal turbinates of rats exposed to 3 ppm of EDB and no lesions attributed to the exposure. Male rats exposed to 40 ppm EDB had an increased incidence of slight focal atrophy of the renal tubules. This effect was not observed in rats necropsied after 1 or 13 weeks of exposure to EDB. In fact, after 1 week of exposure to EDB a decrease in the tubular atrophy of the kidneys was observed from the control group. No other histopathologic observations were considered to be related to exposure to ethylene dibromide.

- 95-96 Day Sacrifice:
Five of ten male rats exposed to 40 ppm of EDB showed a decreased carcass size at the time of necropsy. In addition, five of ten female rats exposed to 40 ppm EDB showed very slight to slight diffuse paleness of the liver. No other grossly visible effects attributable to ethylene dibromide were observed.

Effects considered to be related to EDB exposure were primarily limited to the most anterior sections of the nasal turbinates.

All male and female rats exposed to 40 ppm EDB showed very slight to slight diffuse or focal nonkeratinizing squamous metaplasia and hyperplasia of the respiratory epithelium. In addition, all male and most female rats exposed to 40 ppm showed very slight focal individual epithelial cell necrosis of the respiratory epithelium. Nine of ten rats of each sex exposed to 10 ppm showed very slight to slight degrees of isolated to multifocal hyperplasia of the respiratory epithelium. One female rat of this exposure level showed very slight focal individual epithelial cell necrosis of the respiratory epithelium. The nasal turbinates of rats exposed to 3 ppm exhibited no lesions due to the exposure to ethylene dibromide. The increased incidence of subpleural mononuclear aggregates observed in the lung of rats exposed to EDB occurs at a highly variable rate in this strain of rats in this laboratory and is considered not toxicologically significant.

In view of the grossly observed very slight to slight degree of hepatic paleness, in 5 of 10 females, and the presence of hepatocellular cytoplasmic
vacuolation in H&E stained sections of livers of 2 of 10 females exposed to 40 ppm EDB, Oil Red 0 stained liver sections from all female rats exposed to 0 or 40 ppm EDB were also examined. This revealed a very siight increase of fat within the liver sections of females exposed to 40 ppm EDB. Due to a lack of any significant increase of fat in the livers of females exposed to 40 ppm of EDB, and the absence of any grossly visible hepatic paleness at lower levels, tissues from the lower exposure groups were not stained with Oil Red 0 Stain.

- 88-89 Day Recovery Sacrifice:
One female each from the 3 and 10 ppm groups died spontaneously during the recovery period. Necropsy of these two female rats showed a generalized acute bacterial septicemia, with bacterial organisms noted in various organs and tissues. These deaths were not considered to be related to previous exposure to vapors of EDB. There were no gross pathologic observations which were considered to be related to exposure.

Examination of the sections of the nasal turbinates revealed no evidence of progression of the epithelial hyperplasia or metaplasia that had been previously noted at the 1, 6 or 13 week sacrifices. The nasal turbinates of all 10 males previously exposed to 10 40 ppm ethylene dibromide had no discernible changes in comparison to controls as a result of the exposure. Of the 10 females previously exposed to 40 ppm EDB, 9 of the feamles had no evidence of hyperplasia, metaplasia or other exposure-related pathologic effects within the nasal turbinates. One female rat exposed to 40 ppm EDB had a single focus of epithelial hyperplasia noted in the respiratory epithelium of the nasal turbinates which was probably the sole remnant from the previous exposure to EDB. Examination of the nasal turbinates of females previously exposed to 10 ppm of EDB revealed no evidence of epithelial hyperplasia that had been noted during the exposure. As with the interim sacrifices, there were no exposure-related observations in the nasal turbinates of male or female rats exposed previously to 3 ppm of EDB.

Effect levels

Dose descriptor:
NOEC
Effect level:
3 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Rats exposed to 3ppm EDB showed no consistent effect in any parameter measured.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Lesions of the nasal turbinates produced by exposure of rats to EDB for 90 days in the present study were reversible and nearly completely so within about the same time span used to produce the effect. In view of these findings and the lack of any observable effect in rats of the 3 ppm exposure group, these data indicate that short-term exposure to EDB would not likely result in any irreversible effects on the upper respiratory tract or other tissues of the body.