Registration Dossier

Diss Factsheets

Toxicological information

Repeated dose toxicity: inhalation

Currently viewing:

Administrative data

sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Report date:
Reference Type:
study report
Report date:
Reference Type:
Isobutyraldehyde administered by inhalation (whole body exposure) for up to 13 weeks or two years was a respiratory tract toxicant but was not carcinogenic in F344/N rats and B6C3FI mice.
Abdo, KM et al.
Bibliographic source:
Toxicol. Sci. 42: 136-151

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): Isobutyraldehyde
- Lot No.: 56-202, E042483
- Batch No.: 02, 03
- NCI No.: C60968
- Test material was stored at 4°C
- Purity: 98.6 - 99.1% (major impurity: isobutyric acid 0.53 - 1.4%)
- Physical state: clear, colorless, nonviscous liquid

Test animals

Details on test animals or test system and environmental conditions:
- Source: Frederick Cancer Research Facility, Maryland
- Age at study initiation: 6 weeks
- Housing: individually, multicompartment stainless steel cages (Allentown Caging, Allentown, New Jersey); Bedding: DACB® Neomycin™ Treated Desorb Cageboard (Shepherd Specialty Papers, Inc., Kalamazoo, MI)
- Diet: ad libitum, Zeigler NIH 07 Open Formula Rat and Mouse Ration (Zeigler Brothers, Inc., Gardners, Pennsylvania)
- Water: tap water, ad libitum
- Acclimation period: 13 days

- Temperature (°C): 20.4 – 21.8
- Humidity (%): 43 - 66
- Air changes (per hr): 12 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Details on inhalation exposure:
The exposure chamber concentrations were generated by bubbling nitrogen gas through a column of the liquid maintained at a constant temperature (44 to 46°C) in a water bath. The bubbler used was a gas wash bottle bubbler (PGC Scientific) with a 90-mm-diameter fritted disc of medium porosity at the bottom. Nitrogen gas was pumped from a reservoir (the original container) through the disc at a constant rate and filtered through approximately 1 L of isobutyraldehyde liquid. An explosion-proof fluid metering pump was used to pump the isobutyraldehyde. The bubbler was continuously refilled via a side tube and pressure stopcock to maintain a constant isobutyraldehyde liquid level in the bubbler. Because isobutyraldehyde reacts with the copper tubing, the system was redesigned. The copper tubing was replaced with stainless steel valves, connections, and tubing, and dilution air was added to the nitrogen-borne isobutyraldehyde vapor immediately above the bubbler to prevent condensation of isobutyraldehyde in the manifold or delivery lines when it cooled to room temperature. The vapor was then further diluted with HEPA- and charcoal-filtered air from intake lines at the top of the chambers. Concentrations of isobutyraldehyde vapor were adjusted for the individual exposure chambers by altering either the nitrogen flow rate, the exposure chamber air flow rate, or the water bath temperature. Isobutyraldehyde vapor was transferred into exposure chambers with fine metering valves.

Inhalation chambers of the Rochester design were used. The total volume for each chamber was 1.15 m³. The chamber ventilation system provided 12 to 15 charcoal- and HEPA-filtered air changes per hour and the internal design of the chamber afforded opportunity for equal exposure to each animal. The flow rate was sufficient to maintain the temperature and humidity, to provide a uniform and reproducible test atmosphere, and to remove ammonia.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The chamber concentrations of isobutyraldehyde were monitored on a Wilkes Model 80 infrared spectrophotometer. Samples were drawn and analyzed from each exposure chamber, the control chamber, and the exposure suite 6 to 14 times per exposure period. Samples were drawn through Teflon® tubing.

Generator reservoir and exposure chamber samples were monitored for isobutyric acid by gas chromatography. By determination of peak area ratios, the average ratio of isobutyric acid to isobutyraldehyde was 1.16% pregeneration and 2.54% postgeneration. Gas chromatography of postgeneration isobutyraldehyde samples with an internal standard of isobutyric acid revealed an isobutyric acid content of 7% to 12% in the reservoir. Chamber samples drawn on 2 days had no measurable amount of acid (less than 0.4% to 0.6% isobutyric acid/aldehyde).
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 h/day, 5 days/week
Doses / concentrationsopen allclose all
Doses / Concentrations:
500, 1000, 2000, 4000 and 8000 ppm
other: target conc.
Doses / Concentrations:
504, 994 , 2016, 4034, 8295 ppm (approx. 1.5, 2.9, 5.9, 11.9, 24.4 mg/L)
analytical conc.
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Details on study design:
Based on a 14-day repeated dose toxicity study.


Observations and examinations performed and frequency:
Observed twice daily and clinical findings were recorded weekly

Animals were weighed initially, weekly, and at the end of the study.

Blood was collected by cardiac puncture from all animals surviving the end of the study.
- Parameters examined: hematocrit, hemoglobin concentration, erythrocyte and reticulocyte counts, and total leukocyte counts and differentials

Blood was collected by cardiac puncture from all animals surviving the end of the study.
- Parameters examined: urea nitrogen, alanine aminotransferase, aspartate aminotransferase, and sorbitol dehydrogenase
Sacrifice and pathology:
The brain, heart, right kidney, liver, lungs, right testis, and thymus were weighed.

The following tissues were examined during the gross necropsy: gross lesions, skin, mandibular lymph node, mammary glands, thigh muscle, sciatic nerve, sternum (including marrow), pancreas, spleen, kidneys, adrenals, urinary bladder, seminal vesicles, prostate, testes/epididymides/vaginal tunics of the testes and scrotal sac, esophagus, stomach, duodenum, tissue masses or suspect tumors and regional lymph nodes, ileum, colon, cecum, rectum, mesenteric lymph node, liver, costochondral junction (rib), thymus, oral cavity, larynx and pharynx, trachea, lungs and bronchi, heart and aorta, thyroid, parathyroids, ovaries, uterus, nasal cavity and nasal turbinates, jejunum, tongue, brain, pituitary, spinal cord, preputial or clitoral glands, eyes, Zymbal's glands (auditory sebaceous glands), vagina.

Complete histopathology was performed on 0 and 2,000 ppm mice, and all intercurrent deaths.
Other examinations:
At the end of the studies, sperm samples were collected from all male animals in the 0, 500, 1,000, 2,000, and 4,000 ppm exposure groups for sperm motility and morphology evaluations. The following parameters were evaluated: sperm concentration, motility, and percent abnormality. The right cauda, right epididymis, and right testis were weighed. Vaginal samples were collected for up to 12 consecutive days prior to the end of the studies from all females exposed to 0, 500, 1,000, or 2,000 ppm for vaginal cytology evaluations. The following parameters were evaluated: the relative frequency of estrous stages and estrous cycle length.
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. Animals found dead of other than natural causes or missing were censored from the survival analyses; animals dying from natural causes were not censored. Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The most prominent clinical signs of toxicity (decreased activity, slowed or labored respiration) were observed in males and females at the two highest dose levels, both of which were lethal to all except a single animal.
mortality observed, treatment-related
Description (incidence):
Mortality was 20/20 at 8000 ppm, 19/20 (9/10 males and 10/10 females) at 4000 ppm and 1/10 males each at 1000 ppm and at 0 ppm. The single death in the 1000ppm group is not considered to be related to treatment.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Final mean body weights and body weight gains of male mice were similar to those of the chamber controls. The final mean body weight and body weight gain of female mice in the 1,000 ppm group were slightly but significantly less than those of the chamber controls. Because of the minimal difference and the lack of a dose response, this finding is considered incidental.
female b.w. (g): control 25.9; 500ppm: 24.9; 1000ppm: 24.0; 2000ppm: 25.4
Haematological findings:
no effects observed
Description (incidence and severity):
No exposure-related hematology changes occurred.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute and relative kidney weights of males in the 1,000 and 2,000 ppm groups were significantly greater than those of the chamber controls, though there was no clear dose-response. The absolute liver weight of 1,000 ppm females and absolute and relative liver weights of 500 ppm females were significantly less than those of the chamber controls. These changes only occured in single dose groups and lacked dose dependency. They are thus considered incidental. The absolute thymus weight of 1,000 ppm females and the absolute and relative thymus weights of 2,000 ppm females were significantly less than those of the chamber controls. Considering the local effects on the respiratory tract and the abscence of histopathological findings in the thymus, this change is likely a stress reaction.
Gross pathological findings:
no effects observed
Description (incidence and severity):
At necropsy no gross lesions that could be associated with isobutyraldehyde exposure were observed.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Increased incidences of non-neoplastic lesions of the nasal cavity were observed in mice exposed to 1000 and 2000 ppm isobutyraldehyde. These lesions included necrosis, inflammation, hyperplasia, and squamous metaplasia of the epithelium. Serous and suppurative exudates within the nasal passages was also observed. Mild osteodystrophy of the bones of the maxillo- and nasoturbinates was also observed characterized by decreased number of osteoblasts, increased numbers of osteoclasts, decreased bone density, and increased amounts of periosteal connective tissue. These changes were accompanied by degeneration of the olfactory epithelium characterized by reduced thickness and loss of sensory nuclei.
Mild to moderate lymphoid depletion and/or necrosis occured in the thymus of males and females exposed to 8000ppm only, a concentration, which was deadly to all animals.
Description (incidence and severity):
No differences in the epididymal spermatozoal parameters or estrous cycle lengths were observed in exposed mice.

Effect levels

open allclose all
Dose descriptor:
Effect level:
1.5 mg/L air (analytical)
Based on:
test mat.
Basis for effect level:
other: irritativ lesions of the respiratory tract
Dose descriptor:
Effect level:
5.9 mg/L air (analytical)
Based on:
test mat.
Basis for effect level:

Target system / organ toxicity

Critical effects observed:

Applicant's summary and conclusion