Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP, guidline study; However, rats were exposed for only 28-47 days. Note- This summary only describes procedures and results related to repeated dose toxicity. Reproductive and developmental data are discussed in Sections 5.8.1 and 5.8.2, respectively.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Rats were exposed for only 28-47 days.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
A final purity of 99.69% +/- 0.01% [determined by gas chromatography with thermal conductivity (GC/TCD)] corrected for water, was reported for the sample. The water content of the sample was 0.05%. Infrared spectroscopy (FTIR) and gas chromatographic mass spectroscopy (GC/MS) were used to confirm the proposed structure.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Animals: CD rats (Crl: CD(SD) IGSBR) were approximately eight weeks when treatment was initiated. Examinations performed on all animals prior to the study start revealed that all animals were in good health for study purposes. The animals were acclimated to the laboratory for at least one week prior to the start of the study. Animals had free access to food and water (except during exposure, when both were withheld). Food and water had no contaminants at levels that would interfere with the conduct of this study or interpretation of the results.

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: No data
Details on inhalation exposure:
Test material atmosphere: Exposure chambers were 2 cubic meter stainless steel and glass Rochester-type whole-body exposure chambers [1.3 meters (m) x 1.2 m wide x 1.2 m deep with a pyramidal top and bottom]. The various concentrations of 1-nitropropane were generated using the glass J-tube method (Miller et al., Am Ind Hyg Assoc J, 41:84-846,1980). Liquid test material was pumped into the glass J-tube assemblies (1 per exposure chamber) and vaporized by the flow of nitrogen gas passing through the bead bed of the glass J-tube. The nitrogen was heated as needed with a flameless heat torch to the minimum extent necessary to vaporize the test material. All chambers (including the filtered air control) received the same amount (20 liters per minute) of supplemental nitrogen. The minimum amount of nitrogen necessary to reach the desired chamber concentrations was used. The generation system was electrically grounded and the J-tubes were changed as needed. The vaporized test material and carrier gas were mixed and diluted with supply air to achieve a total flow of 450 liters per minute at the desired test chamber concentration. This flow rate was sufficient to provide the normal concentration of oxygen to the animals and 12-15 calculated air changes per hour. The chamber temperature and relative humidity were controlled by a system designed to maintain values of approximately 22 +/- 3 degrees C and 30 to 70%, respectively. The chambers were operated at a slightly negative pressure, relative to the surrounding area.


Mean chamber concentration values for the study were 0, 24.4 +/- 1.8, 48.4 +/- 1.8, and 96.3 +/- 2.6 ppm. Actual mean chamber concentration values deviated 2-4% from the targeted values of 0, 25, 50, and 100 ppm. Chamber airflow in all four chambers was maintained throughout the study duration. Mean chamber temperatures for all chambers were maintained between 20 and 23 C. Mean chamber relative humidity was maintained in the range of 33-44% for all exposure chambers. On one exposure day the mean relative humidity of the 25, 50, and 100 ppm chambers was below 30% (29.4, 29.4, and 28.1%, respectively). This single, minor excursion from the protocol-specified range of 30-70% is not considered to have affected the integrity of the study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The chamber concentrations of 1-nitropropane, measured approximately in the center of the breathing zone of the animals, were determined at least once per hour with a Miran 1A infrared (IR) spectrophotometer and reported by a strip chart recorder. The IR spectrophotometer was calibrated and a standard curve was compiled prior to and at the midpoint of the study, using air standards prepared by vaporizing measured volumes of 1-nitropropane into Tedlar sample bags along with the metered volumes of dry, compressed air. Analytical concentrations during the exposures were interpolated using the standard curve. The analytical system was checked prior to each exposure with a 1-nitropropane standard gasbag of known concentration. The nominal concentration of the test material in each chamber was estimated based on the amount of test material used and the total airflow through the chamber. Prior to the start of the study, each of the chambers was checked to ensure that a uniform distribution of vapor was present throughout the breathing zone of the animals.
Duration of treatment / exposure:
The females were exposed daily for approximately two weeks prior to breeding, continuing through breeding (two weeks) and continuing through gestation day 19. The males were exposed beginning approximately two weeks prior to breeding and continuing through breeding (two weeks) for a minimum exposure period of 28 days.
Frequency of treatment:
6 hours/day, 7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 25, 50, or 100 ppm vaporized 1-nitropropane
Basis:
nominal conc.
No. of animals per sex per dose:
12/sex/exposure
Control animals:
yes, sham-exposed
Details on study design:
Study conduct: Groups of 12 male and 12 female CD rats were whole-body exposed to target concentrations of 0, 25, 50, or 100 ppm vaporized 1-nitropropane for six hours/day, seven days per week. The concentrations used were based on the results of a previous range-finding study. The females were exposed daily for approximately two weeks prior to breeding, continuing through breeding (two weeks) and continuing through gestation day 19. The males were exposed beginning approximately two weeks prior to breeding and continuing through breeding (two weeks) for a minimum exposure period of 28 days.

Positive control:
No

Examinations

Observations and examinations performed and frequency:
Once each day, a clinical examination was conducted at approximately the same time each afternoon following exposure. This examination included a careful hand-held evaluation of skin, respiration, nervous system function (including tremors and convulsions), swelling, masses, and animal behavior. Cage-side examinations was conducted twice daily and the following parameters were evaluated (if possible): skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), animal behavior, moribundity, mortality, and the availability of feed and water. Detailed clinical observations (DCO) were conducted on all rats pre-exposure, and then weekly throughout the study. Mated females were given detailed examinations on gestation days 0, 7, 14, and 20.

Functional tests (sensory evaluation, rectal temperature, grip performance, and motor activity) were conducted pre-exposure and during the last week of the treatment period. For male rats, this took place on test day 26. For female rats, this took place on lactation day (LD) 4. The sensory evaluation included a test for nociception (responsiveness to tail pinch) and for startle response (responsiveness to sharp noise). The evaluation was conducted in a clear plastic box. Rectal temperature was measured by carefully placing a rectal thermistor approximately 4 cm into the rectum for about 10 seconds. The thermistor was validated at 37 C before and after the study. Hind-limb and forelimb grip performance were tested according to the procedure described by Mattsson et al. (Fund Appl Toxicol 6:175-181, 1986). An automated system was used for motor activity (MA) data collection. No entry into the MA test room was allowed during the testing period. Each test session consisted of ten 8-minute epochs, totaling 80 minutes of testing per animal per test session. This duration was chosen based on the results of a validation study indicating that performance of control animals approached asymptote in 70 - 80 minutes in CD rats. Activity counts for each epoch were recorded.

All rats were weighed at least once during the pre-exposure period and on the first day of exposure. Body weights for males were recorded weekly throughout the course of the study. Females were weighed weekly during the premating and mating periods. During gestation, females were weighed on days 0, 7, 14, and 20. Females that delivered litters were weighed on days 1 and 4 post-partum. Females that failed to mate or deliver a litter were not weighed during the gestation or lactation phases. Statistical analyses of body weights and body weight gains during gestation were performed using data collected on days 0, 7, 14, and 20. Statistical analyses of body weight and body weight gains during the post-partum period were performed using data collected on days 1 and 4.

For males and females, feed consumption was determined weekly during the two week pre-breeding period by weighing feed containers at the start and end of a measurement cycle. During breeding, feed consumption was not measured in males or females due to co-housing. Following breeding, feed consumption was not measured for males. During gestation, feed consumption was measured for females on days 0-7, 7-14, and 14-20. After parturition, feed consumption was measured on LD 1 and 4. Feed consumption was not recorded for females that failed to mate or deliver a litter.

A timed urine volume was obtained from all male rats in each dose group (nonfasted) during the week prior to necropsy (test day 28). Animals were housed in metabolism cages and urine was collected overnight (approximately 16 hours). Urine was analyzed for color, appearance, specific gravity, pH, bilirubin, glucose, proteins, ketones, blood and urobilinogen.

On the day prior to the scheduled necropsy, all males and females in each dose group were fasted overnight. At necropsy, the animals were anesthetized with CO2, and the blood samples were collected from the orbital sinus. Blood samples were not obtained from females that failed to deliver a litter. Blood for hematological analyses was mixed with ethylenediamine-tetraacetic acid (EDTA. Blood smears were prepared, stained with Wright's stain and evaluated using a Hematology Analyzer. Parameters measured were hematocrit (HCT), hemoglobin (HgB) concentration, red blood cell (RBC) count, total and differential white blood cell (WBC) count, platelet (PLAT) count, differential WBC count, red blood cell indices (mean cell hemoglobin concentration, mean cell hemoglobin and mean cell volume) and methemoglobin (MetHgB). Blood for clinical chemistry analyses was collected collected in sodium citrate tubes. Prothrombin time was determined. Serum was analyzed for alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), cholesterol (CHOL), creatinine (CREAT), electrolytes (Na, K, PO4, Cl, and Ca), glucose (GLUC), total bilirubin (TBIL), total protein (TP) and urea nitrogen (UN).
Sacrifice and pathology:
Males were euthanized on test day 29, while females that delivered litters were euthanized on post-partum day 5. Females that did not deliver a litter were euthanized at least 24 days after the last day of the mating period.
Necropsies were conducted by a veterinary pathologist assisted by a team of trained individuals. The necropsy included an examination of the external tissues, and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary, and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The nasal cavity was flushed via the nasopharyngeal duct, and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10% formalin using a hand-held syringe and blunt needle. The thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised.
The following tissues were trimmed and weighed: testes, epididymides, ovaries, liver, kidneys, adrenals, thymus, spleen, brain, thyroid/parathyroid (after fixation), and heart. The organ to body weight ratios were calculated. Similar necropsy procedures were followed for animals found dead or moribund with the exception that terminal body weights and organ weights were not collected.

Representative samples of tissues [adrenals, aorta, auditory sebaceous glands, bone (including joint), bone marrow, brain (cerebrum, brainstem and cerebellum), cecum, cervix, coagulating glands, colon, cranial nerve (optic), duodenum, epididymides, esophagus, eyes, gross lesions, heart, ileum (with Peyer's Patch), jejunum, kidneys, lacrimal/Harderian glands, larynx, liver, lungs, mammary gland, mediastinal lymph node, mediastinal tissues, mesenteric lymph node, mestenteric tissues, nasal tissues/pharynx, oral tissues, ovaries, oviducts, pancreas, parathroid glands, peripheral nerve (tibial), pituitary, prostate, rectum, salivary glands, seminal vesicles, skeletal muscle, skin and subcutis, spinal acord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thryoid gland, tongue, trachea, urinary bladder, uterus and vagina] were collected and preserved in neutral, phosphate-buffered 10% formalin, except that the testes and epididymides were preserved by immersion in Bouin's fixative. Histopathologic examination of these tissues was conducted on all adult rats from the control and high-dose groups. Examination of tissues from the remaining groups was limited to nasal tissues/pharynx and relevant gross lesions. Selected histopathologic findings were graded to reflect the severity of specific lesions to evaluate: 1) the contribution of a specific lesion to the health status of an animal, 2) exacerbation of common naturally occurring lesions as a result of the test material, and 3) dose-response relationships for treatment related effects. Very slight and slight grades were used for conditions that were altered from the normal textbook appearance of an organ/tissue, but were of minimal severity and usually with less than 25% involvement of the parenchyma. This type of change was not expected to significantly affect the function of the specific organ/tissue or have a significant effect on the overall health of the animal. A moderate grade was used for conditions that were of sufficient severity and/or extent (up to 50% of the parenchyma) that the function of the organ/tissue may have been adversely affected, but not to the point of organ failure. The health status of the animal may or may not have been affected, depending on the organ/tissue involved, but generally lesions graded as moderate were not life threatening. A severe grade was used for conditions that were extensive enough to cause significant organ/tissue dysfunction or failure. This degree of change in a critical organ/tissue may have been life threatening.
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):
effects observed, treatment-related
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:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
Small increases in female heart weights correlated positively with dose but were interpreted to be of no biologic significance and likely due to chance for the following reasons: lack of statistical significance, lack of similar effect in males, and the absence of gross or histopathologic changes in the heart in either sex at any dose. Additionally, the absolute and relative heart weights for high-dose females (1.022 and 0.376 g, respectively) were within the range of historical controls for four recent studies of similar experimental design conducted by the investigators (range was 0.958-1.047 g and 0.350-0.370 g for absolute and relative female heart weights, respectively).

The NOEC (no observable effect concentration) set by the investigators was 50 ppm. Effects seen in female lungs at 50 ppm were not considered to be related to treatment. Reasons for this conclusion were not stated. Possible reasons are that inflammation of the squamous epithelium also was seen in some control animals, and olfactory cell degeneration was very slight and focal (compared to slight or multifocal in animals exposed to 96 ppm).

Effects at 96 ppm: All animals survived until termination. Clinical and behavioral observations were normal. Feed consumption was reduced during the pre-breeding phase on test days 1-7 and 7-14 in males and on days 1-7 in females. Average body weight of males was decreased (6.9%) on test day 7. There was no effect of exposure on body weight of females. There was no effect of exposure on any functional test or hematological or urinalysis parameter. There were no changes in clinical chemistry (with the exception that males had a slightly higher albumin concentration than controls (3.4 mg/dl versus 3.2 mg/dl in controls). This alteration was interpreted to be reflective of normal biological variation and not an effect of treatment. Males exposed to 96 ppm had higher relative brain and testes weights. There were no treatment related gross pathologic observations. Treatment-related histopathologic effects were noted in the nasal tissues of one male and up to seven females exposed to 96 ppm. The nasal tissue effects consisted of very slight or slight degeneration of the olfactory epithelium, and slight chronic active inflammation of the squamous epithelium. The olfactory degeneration was characterized by thinning and/or a disarray of olfactory epithelial cells located in the anterior portions of the dorsal meatus or the dorsal portions of the nasoturbinates. Other features of olfactory degeneration in some affected animals consisted of accompanying inflammation in the olfactory epithelium and submucosa, and exfoliation of olfactory epithelial cells into the nasal lumen. Inflammation of the nasal squamous epithelium was characterized by accumulations of neutrophils and lymphocytes in the anterior portion of the squamous epithelium of the ventral meatus. Exfoliation of inflammatory cells and keratin was present in some affected animals. The severity of all lesions was graded very slight to slight.

Effects at 48 ppm: All animals survived until termination. Clinical and behavioral observations were normal. There was no significant effect of exposure on food consumption or body weight of males or females. There was no effect of exposure on any functional test or hematological, clinical chemistry or urinalysis parameter. There were no treatment related gross pathologic observations. Treatment-related histopathologic effects were noted in the nasal tissues of a few females exposed to 48 ppm. Very slight focal degeneration of the olfactory epithelium with degeneration
was noted in 2 females and very slight or slight, chronic, active inflammation of the squamous epithelium was noted in one or two females, respectively.

Effects at 24 ppm: All animals survived until termination. Clinical and behavioral observations were normal. There was no significant effect of exposure on food consumption or body weight of males or females. There was no effect of exposure on any functional test or hematological, clinical chemistry or urinalysis parameter. Males had statistically identified lower absolute and relative thymus weights. The alterations in thymus weights were interpreted to not be treatment-related because of the lack of a dose response. There were no treatment related gross pathologic observations. Very slight, chronic, active, multifocal or focal inflammation of the squamous epithelium was noted in one female.

Effects in controls: Very slight, chronic, active, multifocal or focal inflammation of the squamous epithelium was noted in one and two females, respectively.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
48 ppm (analytical)
Dose descriptor:
LOAEL
Effect level:
96 ppm (analytical)
Dose descriptor:
NOEC
Remarks:
general toxicity
Effect level:
50 ppm (nominal)
Sex:
male
Basis for effect level:
other: based on histologic changes in nasal tissues
Dose descriptor:
NOEC
Remarks:
general toxicity
Effect level:
25 ppm (nominal)
Sex:
female
Basis for effect level:
other: based on histologic changes in nasal tissues
Dose descriptor:
NOEC
Remarks:
reproductive effects
Effect level:
50 ppm (nominal)
Basis for effect level:
other: based on differences in litter size between high-dose animals and controls
Dose descriptor:
NOEC
Remarks:
neurologic function
Effect level:
100 ppm (nominal)
Basis for effect level:
other: 100 ppm was the highest concentration tested

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Exposure to 1-nitropropane resulted in treatment-related decreases in body weight in males exposed to 96 ppm. Treatment-related histopathologic effects were noted in the nasal tissues of males exposed to 96 ppm and females exposed to 48 or 96 ppm. The nasal tissue effects consisted of very slight or slight degeneration of the olfactory epithelium, and slight chronic active inflammation of the squamous epithelium.
Executive summary:

None