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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:
11 November 2002 - 12 February 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
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. The study report was conclusive, done to a valid guidelines and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2011

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: F344/N
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: 5 to 6 weeks
- Weight at study initiation: 99 - 102 g (male) and 86 - 92 g (female)
- Fasting period before study: NDA
- Housing: Individually in stainless steel, wire bottom (Lab Products, Inc., Seaford, DE) cages; changed weekly, rotated weekly. Untreated paper cage pan liner (Sheperd Specialty Papers, Kalamazoo, MI); changed daily.
- Diet (e.g. ad libitum): NTP-2000 irradiated pelleted diet (Zeigler Brothers, Inc., Gardners, PA); available ad libitum, except during exposure periods; changed weekly.
- Water (e.g. ad libitum): Tap water (Richland, WA, municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI); available ad libitum.
- Acclimation period: 12 (females) or 13 (males) days
Before the studies began, five male and five female rats were randomly selected for parasite evaluation and gross observation for evidence of disease. Serological analyses were performed on five male and five female sentinel rats during week 1 and five male and five female chamber control rats at the end of the studies.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Temperature: 72° ± 3° F
- Humidity (%): 50% ± 15%
- Air changes (per hr): 15 ± 2/hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light/day

IN-LIFE DATES: From: 11 November (females) or 12 November (males) 2002 To: 11 February (females) or 12 February (males) 2003

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: Not reported
Details on inhalation exposure:
VAPOR GENERATION AND EXPOSURE SYSTEM
A preheater was necessary for the 3-month study. 1-Bromopropane was pumped through a preheater and into a heated glass column filled with glass beads that increased the surface area for vaporization. Heated nitrogen entered the column from below and assisted in vaporizing the chemical while conveying it into a short distribution manifold. Concentration in the manifold was determined by the chemical pump rate and nitrogen flow rate. The pressure in the distribution manifold was kept fixed to ensure constant flow through the manifold and into all chambers as the flow of vapor to each chamber was adjusted.
Metering valves at the manifold controlled flow to each chamber through individual Teflon® delivery lines that carried the vapor from the manifold to three-way exposure valves at the chamber inlets. The exposure valves diverted vapor delivery to exposure chamber exhaust until the generation system was stable and exposures were ready to proceed. To initiate exposure, the chamber exposure valves were rotated to allow the 1-bromopropane vapor to flow to each exposure chamber inlet duct where it was further diluted with filtered, conditioned air to achieve the desired exposure concentration.
The study laboratory designed the inhalation exposure chamber (Harford Systems Division of Lab Products, Inc., Aberdeen, MD) so that uniform vapor concentrations could be maintained throughout the chamber with the catch pans in place. The total active mixing volume of each chamber was 1.7 m3. A small particle detector was used with and without animals in the exposure chambers to ensure that 1-bromopropane vapor, and not aerosol, was produced. No particle counts above the minimum resolvable level (approximately 200 particles/cm3) were detected.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
VAPOR CONCENTRATION MONITORING
Chamber and room concentrations of 1-bromopropane were monitored by an on-line gas chromatograph. Samples were drawn from each exposure chamber approximately every 20 minutes during each 6-hour exposure period using Hastelloy®-C stream-select and gas-sampling valves in a separate, heated valve oven. The sample lines composing each sample loop were made from Teflon® tubing and were connected to the exposure chamber relative humidity sampling lines at a location close to the gas chromatograph. A vacuum regulator maintained a constant vacuum in the sample loop to compensate for variations in sample line pressure. An in-line flow meter between the vacuum regulator and the gas chromatograph allowed digital measurement of sample flow.
The on-line gas chromatograph was checked throughout the day for instrument drift against an on-line standard vapor of 1-bromopropane in nitrogen supplied by a standard generator. The on-line gas chromatograph was recalibrated as required to meet acceptance criteria. Calibration was performed by comparing chamber concentration data to data from grab samples that were collected with activated coconut charcoal gas sampling tubes, extracted with methylene chloride containing 1-bromobutane as an internal standard, and analyzed using an off-line gas chromatograph. Known volumes of chamber atmosphere were sampled at a constant flow rate ensured by a calibrated critical orifice. The off-line gas chromatograph was calibrated with gravimetrically prepared standards of 1-bromopropane containing 1-bromobutane as an internal standard in methylene chloride.

CHAMBER ATMOSPHERE CHARACTERIZATION
Buildup and decay rates for chamber vapor concentrations were determined with and without animals present in the chambers. At a chamber airflow rate of 15 air changes per hour, the theoretical value for the time to achieve 90% of the target concentration after the beginning of vapor generation (T90) and the time for the chamber concentration to decay to 10% of the target concentration after vapor generation was terminated (T10) was approximately 12.5 minutes. A T90 value of 10 minutes was selected for the 3-month study.
Evaluations of chamber uniformity and persistence and monitoring for 1-bromopropane degradation impurities were conducted periodically throughout the studies by GC. Chamber uniformity was maintained and no degradation was detected.
Duration of treatment / exposure:
The study was a 3-month repeat dose inhalation study where animals were given full body exposure 5 days a week, for 6 hours per day for 14 weeks.
Animals were exposed for 6 hours and 10 minutes, the 10 minute period at the beginning, being the time for the vapour concentration to reach at least 90% of the target concentration
Frequency of treatment:
6 hours per day, 5 days per week, for 14 weeks.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
62.5 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
125 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
250 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
500 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1000 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
10 male, 10 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on decreased body weights and indications of neurotoxicity at 2,000 ppm in the 2-week study, 1-bromopropane exposure concentrations selected for the 3-month inhalation study in rats were 62.5, 125, 250, 500, and 1,000 ppm.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: NDA
- Post-exposure recovery period in satellite groups: NDA
- Section schedule rationale (if not random): Random
Groups of 10 male and 10 female rats were exposed to 1-bromopropane vapor at concentrations of 0, 62.5, 125, 250, 500, or 1,000 ppm, 6 hours plus T90 (10 minutes) per day, 5 days per week for 14 weeks. Additional clinical pathology groups of 10 male and 10 female rats were exposed to the same concentrations for 23 days. One additional exposure day was scheduled during the last exposure week to give the rats at least 2 consecutive days of exposure before terminal sacrifice.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
Clinical findings were recorded on day 9 (male rats) or 10 and then weekly. Core study animals were weighed initially, on day 9 (male rats) or 10, weekly thereafter, and at the end of the studies.
Animals were anesthetized with carbon dioxide, and blood was collected from the retroorbital sinus of clinical pathology rats on days 3 and 23 and from core study rats at the end of the study for hematology and clinical chemistry analyses. Blood samples for hematology analyses were placed in tubes containing potassium EDTA. Erythrocyte, leukocyte, and platelet counts; hemoglobin; packed red cell volume; mean cell volume; mean cell hemoglobin; and mean cell hemoglobin concentration were determined using an Abbott Cell-Dyn 3700 hematology analyzer (Abbott Diagnostic Systems, Abbott Park, IL). Manual hematocrit values were determined using a microcentrifuge (Heraeus Haemofuge, Germany) and a Damon/IEC capillary reader (International Equipment Company, Needham Heights, MA) for comparison to Cell-Dyn values for packed cell volume. Blood smears for rats were stained with Romanowsky-type aqueous stain in a Wescor 7100 Aerospray Slide Stainer (Wescor, Inc., Logan, UT). Leukocyte differential counts for rats were based on classifying a minimum of 100 white cells. Reticulocytes were stained with New Methylene Blue and enumerated as a reticulocyte:erythrocyte ratio using the Miller disc method (Brecher and Schneiderman, 1950). Blood samples for clinical chemistry analyses were placed in tubes containing separator gel and allowed to clot. After clot retraction occurred, the samples were centrifuged, and the serum was aliquoted for assay of serum chemistry analytes using a Roche Hitachi 912 (Roche Diagnostic Corporation, Indianapolis, IN).

Hematology parameters analysed: hematocrit; packed red cell volume; hemoglobin; erythrocyte, nucleated erythrocyte, reticulocyte, and platelet counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte count and differentials

Clinical chemistry parameters analysed: urea nitrogen, creatinine, total protein, albumin, globulin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile acids
Sacrifice and pathology:
At the end of the study the rats were sacrificed by carbon dioxide asphyxiation and samples were collected for sperm motility and vaginal cytology evaluations on rats exposed to 0, 250, 500, or 1,000 ppm. For 12 consecutive days prior to scheduled terminal sacrifice, the vaginal vaults of the females were moistened with saline, if necessary, and samples of vaginal fluid and cells were stained. Relative numbers of leukocytes, nucleated epithelial cells, and large squamous epithelial cells were determined and used to ascertain estrous cycle stage (i.e., diestrus, proestrus, estrus, and metestrus). Male animals were evaluated for sperm count and motility. The left testis and left epididymis were isolated and weighed. The tail of the epididymis (cauda epididymis) was then removed from the epididymal body (corpus epididymis) and weighed. Test yolk was applied to slides and a small incision was made at the distal border of the cauda epididymis. The sperm effluxing from the incision were dispersed in the buffer on the slides, and the numbers of motile and nonmotile spermatozoa were counted for five fields per slide by two observers. Following completion of sperm motility estimates, each left cauda epididymis was placed in buffered saline solution. Caudae were finely minced, and the tissue was incubated in the saline solution and then heat fixed at 65° C. Sperm density was then determined microscopically with the aid of a hemacytometer. To quantify spermatogenesis, the testicular spermatid head count was determined by removing the tunica albuginea and homogenizing the left testis in phosphate-buffered saline containing 10% dimethyl sulfoxide. Homogenization-resistant spermatid nuclei were counted with a hemacytometer.
Necropsies were performed on all core study animals. The heart, right kidney, liver, lung, spleen, right testis, and thymus were weighed. Tissues for microscopic examination were fixed and preserved in 10% neutral buffered formalin (eyes were fixed in Davidson’s solution for up to 3 days and then transferred to 10% neutral buffered formalin), processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 μm, and stained with hematoxylin and eosin.
Complete histopathology was performed on 0 and 1,000 ppm core study rats. In addition to gross lesions and tissue masses, the following tissues were examined to a no-effect level: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, eyes, Harderian gland, heart and aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lung, lymph nodes (mandibular, mesenteric, bronchial, mediastinal), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus.
In addition, the liver, larynx, lung and nose of all rats and the prostate gland of male rats were examined in the remaining groups.
Other examinations:
None
Statistics:
See below

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
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 examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
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:
no effects observed

Effect levels

open allclose all
Dose descriptor:
NOAEC
Effect level:
125 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Decreased sperm motility and hepatovellular cytoplasmic vacuolation at 250 ppm air (nominal concentration)
Dose descriptor:
NOAEC
Effect level:
250 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Hepatocellular cytoplasmic vacuolation at 500 ppm air (nominal concentration)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

All rats survived to the end of the study. The final mean body weight and mean body weight gain of 1,000 ppm males were significantly less than those of the chamber controls. Mean body weights of exposed females were similar to those of the chamber controls.

There were no changes in hematology endpoints that were considered related 1-bromopropane exposure. There were early, transient decreases in albumin and total protein concentrations and alanine aminotransferase activities in most exposed groups of male and female rats. These transient decreases may have been associated with effects of 1-bromopropane on hepatic protein metabolism. Additionally, sorbitol dehydrogenase (SDH) activity was increased at day 23 and at the end of the study in females exposed to 1,000 ppm and at the end of the study in males exposed to 500 or 1,000 ppm. Increased SDH activity would be consistent with mild hepatotoxicity caused by 1-bromopropane.

The absolute and relative liver weights of males exposed to 250 ppm or greater and females exposed to 125 ppm or greater were significantly increased. The absolute spleen weights of females exposed to 125 ppm or greater and the relative spleen weight of 1,000 ppm females were greater than those of the chamber controls. In addition, the absolute and relative right kidney weights of 1,000 ppm females were greater than those of the chamber controls.

There were significant exposure concentration-related decreases in sperm motility in male rats exposed to 250 ppm or greater (6.7%, 10.1%, and 27.7% in the 250, 500, and 1,000 ppm groups, respectively), and there were significant decreases in the number of sperm per gram cauda and the total sperm per cauda (25.2% and 36.8%, respectively), as well as significant decreases in the absolute weights of the cauda (14%) and left epididymis (19%) of 1,000 ppm male rats. Female rats in each of the exposure groups evaluated differed significantly from the chamber controls in the relative amount of time spent in the various estrous cycle stages, with each exposed group spending significantly more time in extended estrus and significantly less time in extended diestrus.

Treatment-related lesions occurred in the liver. There were significantly increased incidences of hepatocellular cytoplasmic vacuolization in males exposed to 250 ppm or greater and in females exposed to 500 or 1,000 ppm. The incidence of hepatocellular vacuolar degeneration was significantly increased in 1,000 ppm females. Hepatocellular cytoplasmic vacuolization consisted of swollen hepatocytes with centrally located nuclei and one to three variably sized vacuoles displacing the cytoplasm. Hepatocellular vacuolar degeneration consisted of a distinct population of enlarged, pale-staining, degenerative, “balloon-like” cells admixed with low numbers of necrotic hepatocytes. The lesion involved hepatocytes surrounding the central vein, but in the most severe cases, the vacuolated hepatocytes extended into the midzonal region.

The incidence of minimal, suppurative, prostatic inflammation in 1,000 ppm males was increased, but the increase was not statistically significant. The increasing trend of this lesion, however, was significant. Because this lesion is a common background finding in F344/N rats, the biological significance of the increased incidence in the 1,000 ppm males is unclear.

Applicant's summary and conclusion

Conclusions:
In the 3-month study, there were no treatment-related deaths and significantly lower body weights occurred only in the 1,000 ppm male rat group.
A mild cytoplasmic vacuolization was present in the liver of all 500 and 1,000 ppm rats, and in half of the 250 ppm male rats. Other indications of mild hepatotoxicity in exposed rats were increased liver weights and increases in serum sorbitol dehydrogenase levels.
There were no exposure-related nasal lesions in rats exposed for 3 months.
Executive summary:

Groups of 10 male and 10 female rats were exposed to 1-bromopropane vapor at concentrations of 0, 62.5, 125, 250, 500, or 1,000 ppm, 6 hours plus T90 (10 minutes) per day, 5 days per week for 14 weeks. Additional clinical pathology groups of 10 male and 10 female rats were exposed to the same concentrations for 23 days. All rats survived to the end of the study. Mean body weights of 1,000 ppm males were significantly less than those of the chamber controls. The increases in sorbitol dehydrogenase activities in 500 ppm males and 1,000 ppm males and females were consistent with the histopathologic evidence of mild hepatotoxicity caused by 1-bromopropane. Liver weights of males exposed to 250 ppm or greater and of females exposed to 125 ppm or greater were significantly increased. Spleen and kidney weights of 1,000 ppm females were significantly increased. Results of sperm count and vaginal cytology evaluations showed exposure concentration-related decreases in sperm motility and counts in male rats, reaching 28% and 37%, respectively, in the 1,000 ppm group. Female rats in all three exposure groups evaluated exhibited altered estrous cycles, spending significantly more time in extended estrus and less time in extended diestrus. The incidences of cytoplasmic vacuolization of the liver were significantly increased in males exposed to 250 ppm or greater and in females exposed to 500 ppm or greater. Hepatocyte degeneration was also observed in 1,000 ppm females.