Isobutyraldehyde

Endpoint summary

Description of key information

No systemic effects were observed in rats and mice after inhalation exposure for 90 days at all concentrations, which did not cause lethality due to lung lesions as a result of a local irritant reaction. The NOAEC in both species was 5.9mg/L (2000ppm).

The mouse was more susceptible to local irritation. The NOAEC in the mouse was 1.5mg/L (500ppm) vs 2.9mg/L (1000ppm) in the rat.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- 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

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

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

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:

yes

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

Remarks:

Doses / Concentrations:
500, 1000, 2000, 4000 and 8000 ppm
Basis:
other: target conc.

Remarks:

Doses / Concentrations:
504, 994 , 2016, 4034, 8295 ppm (approx. 1.5, 2.9, 5.9, 11.9, 24.4 mg/L)
Basis:
analytical conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

DOSE RATIONALE
Based on a 14-day repeated dose toxicity study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS
Observed twice daily and clinical findings were recorded weekly

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

HAEMATOLOGY
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

CLINICAL CHEMISTRY
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:

ORGAN WEIGHTS
The brain, heart, right kidney, liver, lungs, right testis, and thymus were weighed.

GROSS PATHOLOGY
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.

HISTOPATHOLOGY
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.

Statistics:

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.

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:

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.

Dose descriptor:

NOAEC

Remarks:

Local

Effect level:

1.5 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: irritativ lesions of the respiratory tract

Dose descriptor:

NOAEC

Remarks:

Systemic

Effect level:

5.9 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Critical effects observed:

no

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Frederick Cancer Research Facility (Frederick, MD)
- Age at study initiation: 6 weeks
- Quarantine period: 13 days
- Housing: Individually in Stainless steel (Allentown Caging, Allentown, NJ) with DACB® Neomycin™ Treated Desorb Cageboard (Shepherd Specialty Papers, Inc., Kalamazoo, MI)
- Diet: NIH-07 Open Formula pellet diet (Zeigler Brothers, Inc., Gardners, PA); available ad libitum, except during exposure periods, changed daily
- Water: Tap water (Rockville municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI); available ad libitum

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

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

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:

yes

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 hours per day, 5 days per week

Remarks:

Doses / Concentrations:
500, 1000, 2000, 4000, 8000 ppm
Basis:
other: target conc.

Remarks:

Doses / Concentrations:
504, 994 , 2016, 4034, 8295 ppm (approx. 1.5, 2.9, 5.9, 11.9, 24.4 mg/L)
Basis:
analytical conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

DOSE RATIONALE
Based on a 14-day repeated dose toxicity study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS
Observed twice daily and clinical findings were recorded weekly

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

HAEMATOLOGY
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

CLINICAL CHEMISTRY
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:

ORGAN WEIGHTS
The brain, heart, right kidney, liver, lungs, right testis, and thymus were weighed.

GROSS PATHOLOGY
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.

HISTOPATHOLOGY
Complete histopathology was performed on 0 and 2,000 ppm rats, 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, 2,000, or 4,000 ppm for vaginal cytology evaluations. The following parameters were evaluated: the relative frequency of estrous stages and estrous cycle length.

Statistics:

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.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical findings in rats exposed to 4,000 or 8,000 ppm included abnormal respiratory sounds decreased activity, nasal discharge, prostration, and slowed respiration.

Mortality:

mortality observed, treatment-related

Description (incidence):

All rats exposed to 8,000 ppm died before the end of the study. Three male rats and six female rats in the 4,000 ppm groups and one female in the 500 ppm group died before the end of the study. There was no mortality at 1000 and 2000ppm.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The final mean body weight of 4,000 ppm males and the body weight gains of 4,000 ppm males and females were significantly less than those of the chamber controls.

Haematological findings:

no effects observed

Description (incidence and severity):

A minimal mature neutrophilia, evidenced by increased segmented neutrophil numbers, occurred in male and female rats; these findings would be consistent with upper respiratory tract inflammation, but the change was not dose-dependent and did not correlate with histopathological findings. It is thus considered incidental.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Minimal increases in alanine aminotransferase activity occurred in an exposure-related manner in male and female rats. Usually, aspartate aminotransferase is an eralier maker for liver toxicity, but there was no increase in the concentration of this enzyme. Sorbitol dehydrogenase activity, another marker of increased hepatocellular permeability/leakage, also did not increase. Considering the small and likely non-biologically relevant increase (49 IU/l compared to 31 IU/l) and the abscence of any other markers for liver toxicity including histopathological results, this alteration is not considered of toxicological relevance. There were no further differences in hematology and clinical chemistry parameters.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The relative kidney weight of 4,000 ppm males was significantly greater than that of the chamber controls. Absolute liver and thymus weights of male rats in the 4,000 ppm group were significantly less than those of the chamber controls. The absolute brain weight of female rats in the 4,000 ppm group was significantly less than that of the chamber controls. These absolute organ weight differences are likely secondary to body weight differences.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross lesions that could be attributed to exposure of the test substance were not evident at necropsy.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Effects were almost only observed at lethal concentrations: Male and female rats exposed to 8,000 ppm had congestion and severe necrosis of the epithelium, and occasionally of the entire mucosa, of the nasal turbinates accompanied by acute inflammation, and accumulation of serous or fibropurulent exudat within the nasal passages. Male and female rats exposed to 4,000 ppm had mild epithelial hyperplasia of the mucosa of the nasal cavity and nasopharynx. Increased incidences of squamous metaplasia and mild acute (suppurative) inflammation occurred in male and female rats exposed to 4,000 ppm. In addition, male rats exposed to 4,000 or 8,000 ppm and females exposed to 4,000 ppm had mild osteodystrophy in the bones of the maxillo- and nasoturbinates characterized by decreased numbers of osteoblasts, increased numbers of osteoclasts, decreased bone density, and increased amounts of periosteal connective tissue. These changes were accompanied by inflammation of the overlying mucosa. Minimal to mild degeneration of the olfactory epithelium characterized by reduced thickness and loss of sensory cell nuclei was observed in all male rats exposed to 2,000 or 4,000 ppm and in three female rats exposed to 2,000 ppm. In the larynx and trachea, the incidences of necrosis/degeneration were increased in male rats exposed to 8,000 ppm. Mild to moderate lymphoid depletion of the spleen and thymus was seen only in male and female rats exposed to 8,000 ppm and may have been a direct toxic effect and/or due to stress associated with administration of the test substance. The incidences of mild to moderate lymphoid necrosis of the thymus were significantly increased in 8,000 ppm male (10/10) and female (8/10) rats only. Minimal to mild maturation arrest in the testis characterized histologically as decreased numbers of germinal cells, reduced spermatogenesis, and/or increased numbers of large mononuclear cells (immature spermatogonia) in the ducts of the epididymis occurred in 8,000 ppm males (9/10). All 8,000 ppm males died during the first week of exposure.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

The caudae and epididymis weights of 4,000 ppm male rats were significantly less than those of the chamber controls. These decreases are likely secondary to lower mean body weights. Females exposed to 4,000 ppm differed significantly from the chamber control females in the relative time spent in the estrous stages. Generally, these females spent more time in diestrus and less time in proestrus than the chamber control females.
Spermatozoal motility in 500 and 1,000 ppm males, but not of 2000ppm males was significantly reduced as compared to the chamber controls. Due to a lack in dose-response, this change is not considered relevant. There were no further alterations for males or females.
In conclusion, there were no toxicologically relevant differences between treated and control animals at non-lethal concentrations.

Dose descriptor:

NOAEC

Remarks:

Local

Effect level:

2.9 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Effects on the olfactory epithelium

Dose descriptor:

NOAEC

Remarks:

Systemic

Effect level:

5.9 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

5 900 mg/m³

Study duration:

subchronic

Species:

other: Mouse and rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- 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

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

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

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:

yes

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

Remarks:

Doses / Concentrations:
500, 1000, 2000, 4000 and 8000 ppm
Basis:
other: target conc.

Remarks:

Doses / Concentrations:
504, 994 , 2016, 4034, 8295 ppm (approx. 1.5, 2.9, 5.9, 11.9, 24.4 mg/L)
Basis:
analytical conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

DOSE RATIONALE
Based on a 14-day repeated dose toxicity study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS
Observed twice daily and clinical findings were recorded weekly

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

HAEMATOLOGY
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

CLINICAL CHEMISTRY
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:

ORGAN WEIGHTS
The brain, heart, right kidney, liver, lungs, right testis, and thymus were weighed.

GROSS PATHOLOGY
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.

HISTOPATHOLOGY
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.

Statistics:

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.

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:

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.

Dose descriptor:

NOAEC

Remarks:

Local

Effect level:

1.5 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: irritativ lesions of the respiratory tract

Dose descriptor:

NOAEC

Remarks:

Systemic

Effect level:

5.9 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

1 500 mg/m³

Study duration:

subchronic

Species:

mouse

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: inhalation

Rat

In a 90 -day study, groups of 10 male and 10 female F344/N rats were exposed to 0, 500, 1000, 2000, 4000, or 8000 ppm isobutyraldehyde by inhalation, 6 hours per day, 5 days a week, for 13 weeks (NTP 1999). All rats exposed to 8000 ppm died within the first week. Three male rats and six female rats in the 4000 ppm groups and one female in the 500 ppm group also died before the end of the study. Since there was no mortality in intermediate doses, the single death at 500 ppm is considered incidental. The final mean body weight of male rats in the 4000 ppm group and the body weight gains of 4000 ppm males and females were significantly less than those of the chamber controls. Clinical findings in rats exposed to 4000 or 8000 ppm included abnormal respiratory sounds, decreased activity, nasal discharge, prostration, and slowed respiration. In the 8000 ppm groups, severe necrosis of the epithelium, and occasionally of the entire mucosa, of the nasal turbinates accompanied by an acute inflammatory reaction was observed. Increased incidences of squamous metaplasia and mild acute inflammation occurred in male and female rats exposed to 4000 ppm. Minimal to mild degeneration of the olfactory epithelium was observed in all male rats in the 2000 and 4000 ppm groups. Male rats exposed to 4000 or 8000 ppm and females exposed to 4000 ppm had mild osteodystrophy of the turbinate bone. The incidences of necrosis/degeneration of the larynx and trachea were increased in male rats in the 8000 ppm group. The incidences of mild to moderate lymphoid depletion of the spleen and thymus and lymphoid necrosis of the thymus were significantly increased in male and female rats exposed to 8,000 ppm. The NOAEC for male and female rats was determined to be 1000 ppm (ca. 2.9 mg/L air) for local effects and 2000 ppm (ca. 5.9 mg/L air) for systemic effects.

Mice

In the 90 -day study, 10 male and 10 female B6C3F1 mice were exposed to 0, 500, 1000, 2000, 4000, or 8000 ppm isobutyraldehyde by inhalation (NTP 1999), 6 hours per day, 5 days per week, for 13 weeks. One male in the chamber control group, one male in the 1000 ppm group (both considered incidental due to lacking dose response), nine males and all females in the 4000 ppm groups, and all males and females in the 8000 ppm groups died before the end of the study. Clinical findings in the two top doses included decreased activity, tremors, prostration, and slower and labored respiration. There were no gross lesions observed at necropsy that could be associated with isobutyraldehyde exposure. Histopathologically, the nasal cavity and lymphopoietic tissues were considered target organs, with changes similar, but not identical, to those observed in rats. Increased incidences of nonneoplastic lesions of the nasal cavity were observed in male and female mice exposed to 1,000 ppm or greater. These lesions included necrosis, inflammation, hyperplasia, and squamous metaplasia of the epithelium; serous and suppurative exudate within the nasal passages; olfactory epithelial degeneration; and osteodystrophy of the turbinate bone. Mild to moderate lymphoid depletion and/or lymphoid necrosis were observed in the thymus of male and female mice exposed to 8000 ppm. The NOAEC for male and female mice was determined to be 500 ppm (ca. 1.5 mg/L air) for local effects and 2000 ppm (ca. 5.9mg/L) for systemic effects.

Gage et al. (1970) published an article in which four rats per sex were exposed to 1000 ppm isobutyraldehyde 12 times for 6 hours per time. After the exposure period animals were necropsied. Slight nose irritation was observed. No abnormalities were observed upon necropsy.

Justification for classification or non-classification

Based on the available information classification for repeated dose toxicity is not warranted in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.