1,3,3-trimethyl-N-(2-methylpropylidene)-5-[(2-methylpropylidene)amino]cyclohexanemethylamine

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

chronic toxicity: inhalation

Remarks:

combined repeated dose and carcinogenicity

Type of information:

other: supporting study about the hydrolysis product Isobutyraldehyde

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study according to good laboratory practice (GLP)

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1998

Report date:

1997

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Isobutyraldehyde

EC Number:

201-149-6

EC Name:

Isobutyraldehyde

Cas Number:

78-84-2

Molecular formula:

C4H8O

IUPAC Name:

2-methylpropanal

Test material form:

aerosol dispenser: not specified

Remarks:

migrated information: aerosol

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

Isobutyraldehyde was studied by exposing male and female F344/N rats and B6C3F mice

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Details on inhalation exposure:

cited from the Abdo et al. (1998) publication:
Isobutyraldehyde vapor for the 13-week studies was generated by bubbling nitrogen gas through a column of the liquid maintained at a constant temperature in a water bath. The bubbler was continuously refilled via a side stainless steel tube and pressure stopcock to maintain a constant isobutyraldehyde liquid level in the bubbler. Diluting air was added to the nitrogen-borne vapor immediately above the bubbler to prevent condensation of isobutyraldehyde in the mainfold or delivery lines when it cooled to room temperature. 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. Inhalation chambers (1,15 m3 each for teh 13-week and 2,3 m3 for the 2-year studies) of the Rochester design were used in the studies. The chamber ventilation system provided 12 to 15 charcoal- and HEPA-filtered air changes per hour and the internal design of the chamber afforded equal exposure to each animal. This flow rate was sufficient to meintain proper temperature and humidity, provide a uniform and reproducible test atmosphere, and remove ammonia. A small particle detector (Type CN, Gardner Associates, Schenectady, NY) was used with and without animals in the exposure chambers to ensure that isobutyraldehyde vapor , and not aerosol, was produced. No particle counts above the minimum resolvable level (approximately 200 particles/cm2) were detected.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

cited from the Abdo et al. (1998) publication: The chamber concentrations of isobutyraldehyde in the 13-week studies were monitored 6 to 14 times per exposure period on a Wilkes model 80 infrared spectrophotometer. This method determines total aldehyde concentration. A method that specifically measures the chemical being is an NTP prerequisite for the 2-year studies. For this reason chamber concentrations in the 2-year studies were monitored foru times per hour using a 8-port stream select valve with two on-line gas chromatographs.

Duration of treatment / exposure:

Thirteen weeks and 2 years

Frequency of treatment:

Thirteen weeks treatment: 6h per day, 5 days per week
2 years: 6h per day, 5 days per week

Doses / concentrations

Remarks:

Doses / Concentrations:
Thirteen weeks: 0, 500, 1000, 2000, 4000, 8000 ppm; 2 years: 0, 500, 1000, 2000 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

Thirteen weeks: groups of 10 male and female rats and mice per dose
2 years: groups of 50 male and female rats and mice per dose

Control animals:

yes, concurrent no treatment

Details on study design:

cited from Abdo et al. (1998): Four-week old male and female F344/

Positive control:

no data

Examinations

Observations and examinations performed and frequency:

Clinical observations were recorded once per week. Animals were weighed prior to initiation, and then weekly and at the end of the study. Necropsy was performed on all animals. The brain, heart, right kidney, liver, lungs, right testis, and thymus were weighed. Visible lesions and tissues masses were subject to microscopic examination.

Sacrifice and pathology:

Complete histopathologic examination was performed on control and 4000 ppm male rats, and control and 2000 ppm females.
The following tissues were examined during the gross necropsy. A complete gross necropsy is defined as external examination including body orifices and examination and fixation of these tissues: 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.

Other examinations:

Hematology, clinical chemistry, sperm morphology and vaginal cytology evaluation

Statistics:

method used for the evaluation of tumor incidence: logistic regression analysis, life table test, Fisher exact test and Cochran Armitage trend test

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

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

Description (incidence and severity):

Chemical-related nonneoplastic lesions were limited to the nose of rats and mice

Histopathological findings: neoplastic:

no effects observed

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

cited from Abdo et al. (1998):

Thirteen-Week Studies Rats: All rats exposed to 8000 ppm died before the end of the study. Three male rats and six female rats in the 4000 ppm groups and one female in the 500 ppm group died before the end of the study. The final body mean weight of 4000 ppm male and the mean body weight gains of 4000 ppm males and females were significantly less than those of the controls. Clinical findings in rats exposed to 4000 or 8000 ppm included abnormal respiratory sounds, decreased activity, nasal discharge, prostration, and slowed respiration. No biologically significant organ weight changes were observed. Gross lesions that could be attributed to isobutyraldehyde exposure were not evident ant necropsy. Male and female rats exposed to 8000 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 exudate within the nasal passages. Male and female rats exposed to 4000 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 4000 ppm. In addition , male rats exposed to 4000 or 8000 ppm and females exposed to 4000 ppm hat mild osteodystrophy in the bones of the maxillo- and nasoturbinates characterized by decreased numbers of osteoblasts, increased numbers of osteoclasts, decreased bone densitiy, 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 2000 or 4000 ppm and in three female rats exposed to 2000 ppm. In the larynx and trachea , the incidences of necrosis/degeneration were increased in male rats exposed to 8000 ppm.

Mouse study: One male in the control group, one male in the 1000 ppm group, nine males in the 4000 ppm group, and all males in the 8000 ppm group died before the end of the study. All female mice in the 4000 and 8000 ppm groups died before the end of the study. Final mean body weights and mean body weight gains of mal mice were similar to those of the controls. The final mean body weight and mean body weight gain of female mice in the 1000 ppm group were signficantly lower than those of the controls. The absolute and relative kidney weights of males in the 1000 and 2000 ppm groups were significantly greater thatn those of the chamber controls. The absolute liver weight of 1000 ppm females and absolute and relative liver weights of 500 ppm females were signficantly less than those of the chamber controls. The absolute thymus weight of 1000 ppm females and the absolute and relative thymus weight of 2000 ppm females were sgnificantly less than those of the chamber controls. Because of lack of dose response and the absence of lesions related to exposure to isobutyraldhyde in these organs, these variations in organ weights are not considered to be chemically related. There were no gross lesions observed at necropsy that could be associated with isobutyraldehyde exposure. Increased incidences of nonneoplastic lesions of the nasal cavity were observed in male and female mice exposed to 1000 ppm or greater; these lesions were histologically similar to those that occured in rats. These lesions included necrosis, inflammation, hyperplasia, and squamos metaplasia of the epithelium; serous and suppurative exudate within the nasal passages; olfactory epithelial degeneration; and osteodystrophy of the turbinate bone.

Two-Year mouse study: Estimates of 2-year survival probabilities for male and female rats are shown in the Kaplan-Meier survival curves (Fig. 1). There were no significant differences in survival rates between exposed and control male or female rats. The mean body weights of male and female rats were generally similar to those of the controls throughout the study (Fig. 2). No clinical findings that could be attributed to isobutyraldehyde exposure were observed. Pathology. No increase in incidence of neoplasms was observed in rats of either sex that could he attributed to isobutyraldehyde administration (NTP, 1997). However three primary nasal neoplasms were observed in male and female rats exposed to isobutyraldehyde. One polypoid adenoma was present in the anterior nose section of a male rat exposed to 1000 ppm, one adenoma of the vomeronasal organ was noted in a 2000 ppm male, and an undifferentiated malignant neoplasm, classified as a sarcoma (mesenchymal origin), was present in the most posterior section of the nose in a 500 ppm female (Table 3). Exposure-related nonneoplastic lesions in the nose consisted of squamous metaplasia of the respiratory epithelium, degeneration of the olfactory epithelium, and suppurative inflammation. The incidences of minimal to mild squamous metaplasia in 1000 and 2000 ppm males and females and in 500 ppm females were significantly greater than those in the controls (Table 3). This lesion occurred most frequently in the median septum and the medial aspect of the maxillary turbinates and was characterized by replacement of the cuboidal and columnar ciliated epithelium and some keratinization in the surface cells. The incidences of minimal to mild degeneration of the olfactory epithelium in male and female rats exposed to 2000 ppm were significantly greater than those in the controls. Olfactory epithelial degeneration occurred in the dorsal wall in Level II of the nasal cavity. The affected olfactory epithelium had fewer layers of sensory cells and the remaining cells were flattened and irregular (Plates 3 and 4). The incidences of suppurative inflammation (rhinitis) in male and female rats exposed to 2000 ppm were increased compared to the controls. This lesion was most notable in the anterior nasal section and occurred sometimes, but not always, in association with squamous metaplasia of the respiratory epithelium. The inflammation tended to be focal and was occasionally associated with foreign material.

Survival, body weights, and clinical findings. Estimates of 2-year survival probabilities for male and female rats are shown in the Kaplan-Meier survival curves (Fig. 1). There were no significant differences in survival rates between exposed and control male or female rats. The mean body weights of male and female rats were generally similar to those of the controls throughout the study (Fig. 2). No clinical findings that could be attributed to isobutyraldehyde exposure were observed. Pathology. No increase in incidence of neoplasms was observed in rats of either sex that could he attributed to isobutyraldehyde administration (NTP, 1997). However three primary nasal neoplasms were observed in male and female rats exposed to isobutyraldehyde. One polypoid adenoma was present in the anterior nose section of a male rat exposed to 1000 ppm, one adenoma of the vomeronasal organ was noted in a 2000 ppm male, and an undifferentiated malignant neoplasm, classified as a sarcoma (mesenchymal origin), was present in the most posterior section of the nose in a 500 ppm female (Table 3). Exposure-related nonneoplastic lesions in the nose consisted of squamous metaplasia of the respiratory epithelium, degeneration of the olfactory epithelium, and suppurative inflammation. The incidences of minimal to mild squamous metaplasia in 1000 and 2000 ppm males and females and in 500 ppm females were significantly greater than those in the controls (Table 3). This lesion occurred most frequently in the median septum and the medial aspect of the maxillary turbinates and was characterized by replacement of the cuboidal and columnar ciliated epithelium and some keratinization in the surface cells. The incidences of minimal to mild degeneration of the olfactory epithelium in male and female rats exposed to 2000 ppm were significantly greater than those in the controls. Olfactory epithelial degeneration occurred in the dorsal wall in Level II of the nasal cavity. The affected olfactory epithelium had fewer layers of sensory cells and the remaining cells were flattened and irregular (Plates 3 and 4). The incidences of suppurative inflammation (rhinitis) in male and female rats exposed to 2000 ppm were increased compared to the controls. This lesion was most notable in the anterior nasal section and occurred sometimes, but not always, in association with squamous metaplasia of the respiratory epithelium. The inflammation tended to be focal and was occasionally associated with foreign material. Downloaded from http://toxsci.oxfordjournals.org/ by guest on October 16, 2012

Two-Year Mouse Study

Survival, body weights, and clinical findings. Estimates of rates among male mice decreased with increasing exposure 2-year survival probabilities for male and female mice are concentration. The survival rate of males exposed to 2000 ppm was marginally reduced relative to the controls. There were no Mean body weights are given in Fig. 4. The mean body significant differences in survival rates between exposed and weights of male mice were generally similar to those of the control females. controls throughout the study. The mean body weights of female mice exposed to 1000 or 2000 ppm were lower than those of the controls during the second year of the study. No clinical findings that could be attributed to isobutyraldehyde exposure were observed. No increase in tumor incidence was observed in mice (NTP, 1997). The incidences of olfactory epithelial degeneration in 1000 and 2000 ppm males and females were significantly greater than in the controls (Table 4). Degeneration of the olfactory epithelium was minimal to mild in severity and occurred in the dorsal meatus of Level II; in a few mice, Level HI was also involved. Affected olfactory epithelium was characterized by fewer layers of sensory cells, which were often disorganized, and was irregular in thickness. In some mice, only sustentacular and basal cell layers persisted, or ciliated, columnar, respiratory-like epithelium replaced areas of the olfactory epithelium. In some areas, only a thin layer of fusiform cells covered the surface; in others, the basal cells remained. Two 1000 ppm females, one 2000 ppm male, and one 2000 ppm female had necrosis of the olfactory epithelium. Necrotic olfactory epithelium had pyknotic or karyorrhectic nuclei and dense eosinophilic cytoplasm.

Applicant's summary and conclusion

Conclusions:

Isobutyraldehyde administered by inhalation (whole body exposure) for up to thirteen weeks or two years was a respiratory tract toxicant but was not carcinogenic in F344/N Rats and B6C3F, Mice.
NOAEL systemic effects: ca 2.9 mg Isobutyraldehyde/l ; LOAEC: ca 5.8 mg Isobutyraldehyde/l
(based on conversation factor 1 ppm= 2.9 mg/m3, see NTP report TR-472)

Executive summary:

Isobutyraldehyde administered by inhalation (whole body exposure) for up to Thirteen Weeks or Two Years Was a Respiratory Tract Toxicant but Was Not Carcinogenic in F344/N Rats and B6C3F, Mice. Abdo, K. M, Haseman, J. K., and Nyska, A. (1998). Toxicol. Sci. 42, 136-151. Isobutyraldehyde (a chemical structurally related to formaldehyde and used as a flavoring agent) was studied for toxicity and carcinogenicity by exposing male and female F344/N rats and B6C3F, mice. Animals were exposed to isobutyraldehyde vapors 6 h per day, 5 days per week for up to 13 weeks or 2 years. In the 13-week studies, groups of 10 male and 10 female F344/N rats and B6C3F, mice were exposed to concentrations of 0, 500, 1000, 2000, 4000, or 8000 ppm. Chemical-related body weight depression and deaths occurred in rats and mice exposed to 4000 and 8000 ppm. Necrosis of the epithelium accompanied with acute inflammatory reaction was observed in the nasal turbinate, larynx, and trachea of rats exposed to 8000 ppm. Exposure of rats to 4000 ppm resulted in metaplasia of the nasal respiratory epithelium, inflammation, degeneration of the olfactory epithelium, and osteodystrophy of the nasal turbinate bone. In the 13-week mouse study, exposure to 8000 ppm or 4000 ppm resulted in necrosis of the epithelium lining of the nasal turbinates. Osteodystrophy of the nasal turbinate bone and squamous metaplasia of the nasal respiratory epithelium were noted in mice exposed 4000 ppm. Degeneration of the olfactory epithelium was noted in males exposed 2000 ppm and in females exposed to 4000 ppm. In the 2-year studies, groups of 50 male and 50 male F344/ N rats and B6C3F1 were exposed to concentrations isobutyraldehyde vapors of 0, 500, 1000, or 2000 ppm 6 h per day, 5 days per week. There were no differences in survival rates or mean body weights between exposed groups and control rats. Survival of male mice exposed to 2000 ppm and mean body weights of female mice exposed to 1000 or 2000 ppm were lower than those of the of the controls. No increase in neoplasm incidence was observed in rats and mice in the 2-year studies that could be attributed to isobutyraldehyde exposure. Chemical-related nonneoplastic lesions were limited to the nose of rats and mice. They included squamous metaplasia of the respiratory epithelium (rats), suppurative inflammation (rats), and olfactory epithelial degeneration (rats and mice) at 1000 and 2000 ppm.