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Repeated dose toxicity: inhalation

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sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
96 days
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted in a manner similar to OECD 413 and OPPTS 870.3465 guidelines; while there is no indication it was run in accordance with GLPs, it is a well-conducted study that has been published in a peer-reviewed journal

Data source

Referenceopen allclose all

Reference Type:
study report
Report Date:
Reference Type:
Subchronic Inhalation Toxicity of Methyl isoamyl Ketone in Rats
Katz GV, Renner ER Jr. and Terhaar CJ
Bibliographic source:
Fundamental and Applied Toxicology 6, 498-505

Materials and methods

Test guidelineopen allclose all
equivalent or similar to
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
equivalent or similar to
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
Principles of method if other than guideline:
The following are required by the guidelines but were not conducted in the present study: ophthalmological examination and urinalysis.
GLP compliance:
Limit test:

Test material

Details on test material:
-Test substance name (as cited in report): MIAK
-Source of test material: Tennessee Eastman Company, Kingsport, TN
-Purity (analytical report from supplier): 99.1% (Impurities: 0.36% MIBK, 0.28% MAK)
-Purity (performed at test facility): 99.1 and 99.0% (Impurities: 0.34-0.35% 4-methyl-2-pentanone; 0.37-0.38% 2-heptanone; < 0.13-0.14% unidentified)
-Appearance: clear

Test animals

Details on test animals and environmental conditions:
-Strain: CRL:COBS®CD®(SD) BR
-Source of test animals: Charles River Breeding Laboratories, Wilmington, MA
-Weight at study start: males 217-259g, females 139-199g
-Quarantine period: 10 days
-Housing: Singly in stainless steel wire-mesh cages
-Feed: Purina Rodent 5001 Chow, pelleted; ad libitum during non-exposure period
-Water: ad libitum during non-exposure period
-Method of identification: metal ear tag

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
other: Fresh air
Details on inhalation exposure:
Rats were exposed in stainless steel and glass inhalation chambers with an effective volume of 420L. Rats were housed singly in suspended stainless steel wire mesh cages during exposure and were transferred to clean stainless-steel wire mesh cages during non-exposure periods. Four rats were housed doubly during exposure due to caging limitations.

-Animal Rotation:
Animals were rotated daily in a non-random sequential manner according to a numbered cage pattern to compensate for any possible variation in vapor concentrations within the chamber.

-Vapor generation:
Filtered and dried (silica gel) compressed air was metered over the surface of the test substance (2.5L MIAK in a 5.0L bottle). To achieve a concentration of 2000 ppm, the MIAK was heated to 47C. For the 1000 and 200 ppm vapor concentraions, it was heated to 32°C. The vapors were directed to the throat of the inhalation chambers where they were mixed with chamber supply fresh air before entering the chamber. Each test substance generator was replenished with fresh MIAK on a daily basis. On a weekly basis all generators were thoroughly cleaned and all MIAK was replaced.

Chamber concentrations were controlled by metering the generator supplied vapor and fresh air to maintain approximately 23 air changes per hour. Chamber flow was monitored using a calibrated magnehelic gauge measuring the differential pressure across an orifice meter located in each chamber exhaust duct. A negative pressure of 0.25 inches of water was maintained in each chamber.

-Vapor analysis:
Chamber vapor concentrations were monitored and recorded hourly with an automated, multipositional air sampling and analysis system consisting of a Perkin-Elmer Sigma IB Gas chromatograph fitted with a Valco Instruments, 10-port environmental sampling station with an interchangeable 1.0 ml gas sampling loop, and a column switching value. This system operated pneumatically.

-Chamber Temperature and Humidity:
Chamber temperature was recorded hourly. Chamber humidity was also recorded hourly with a modified Taylor hydrometer (Taylor Instruments, Sybron Corp., Arden, NC).
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Purity analysis was performed on two separate occasions by gas chromatographic-mass spectroscopic examinations.

-Nominal chamber concentrations:
Nominal concentrations of 1986±29 ppm, 1011±10 ppm, and 298±35 ppm were calculated by dividing the amount of the test substance vaporized by the total chamber air flow.

-Chamber concentrations:
Concentrations were monitored by gas chromatography. Chamber vapor samples were continuously collected through a Teflon sampling line that was flushed for one minute through the gas sampling loop prior to its injection into the gas chromatograph column. The Perkin-Elmer microprocessor computed sample concentration from the resultant peak areas and sent the data along with all relevant study information to a printer for inclusion in the paper raw data.
Duration of treatment / exposure:
6 hours/day, 5 days/week
Frequency of treatment:
69 inhalation exposures spanning 96 calendar days
Doses / concentrationsopen allclose all
Doses / Concentrations:
200 ppm
other: target concentration
Doses / Concentrations:
1000 ppm
other: target concentration
Doses / Concentrations:
2000 ppm
other: target concentration
No. of animals per sex per dose:
15 rats/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
One hundred twenty rats (60 male and 60 female) were divided into 8 groups of 15 rats each. Rats were exposed to target concentrations of 0, 200, 1000, or 2000 ppm MIAK 6 hours/day, 5 days/week for a total of 69 exposures spanning 96 days.


Observations and examinations performed and frequency:
Clinical Observations:
All rats were observed twice daily (before and after inhalation exposure). Animals that could be observed visually through the chamber windows were observed for signs of toxicity during inhalation exposure.

Body weights:
Individual body weights were determined weekly.

Blood collection and analysis:
Blood was collected from the posterior vena cava under sodium pentobarbital anesthesia. Analyses were performed by the Laboratory Animal Analysis Group, Health, Safety and Human Factors Laboratory, Eastman Kodak Company.

Blood was analyzed for the following parameters: hemoglobin concentration, hematocrit, red blood cell counts, white blood cell counts, differential white blood cell counts, platelet counts, and red cell indices.

Serum Clinical Chemistry:
Blood was analyzed for the following parameters: alanine aminotransferase, aspartate aminotransferase, lactic dehydrogenase, alkaline phosphatase, creatinine, urea nitrogen, and glucose.
Sacrifice and pathology:
Animals were sacrificed between Days 93 and 96 by exsanguination under sodium pentobarbital anesthesia. Daily necropsies were determined by a stratified randomization of animals which resulted in equal representation of group and sex on each day.

Gross Pathology and Organ Weights:
All animals were given a complete necropsy and the following organs were weighed: liver, kidneys, brain, adrenal glands, testes, ovaries, heart, and spleen. Organ weights relative to body weights were subsequently calculated.

The following tissues were fixed in 10% buffered formalin, embedded in paraffin, stained with hematoxylin-eosin and examined by a pathologist: nasal passages, trachea, lungs, thymus, salivary glands, heart, tongue, esophagus, stomach, small intestine, large intestine, liver, kidneys, urinary bladder, adrenal glands, pancreas, thyroids, parathyroids, spleen, mesenteric lymph nodes, bone marrow, brain (medulla oblongata, cerebellum, and cerebral cortex with thalamus and basal ganglia), pituitary glands, testes epididymides, and accessory sex organs in males and fallopian tubes, uterus, vagina, and ovaries in the females. Eyes were fixed in a modified Zenker’s solution and examined.
Numerical data were analyzed by one-way analysis of variance (ANOVA), Bartlett’s test, and Duncan’s multiple range test. A 5% level of significance was used.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
One 1000 ppm male rat died on day 25. There were no gross or histopathological abnormalities and the cause of death could not be determined.

Clinical observations:
During inhalation exposure to 2000 ppm MIAK, both males and females exhibited slight lethargy and a decreased aural response. For the first 17 days of the study, these findings were also present in the 1000 ppm animals. In MIAK-treated males, “gel-like” casts that appeared as translucent rods 3-4 mm long and 1 mm in diameter were observed beneath the cages starting on Day 35; these were present almost daily for the remainder of the study. The significance of the presence of these casts is unclear, but they may represent excretions of seminal fluid and have been seen in males during inhalation studies on other compounds. All other clinical observations that occurred (alopecia, porphyrin-like discoloration, and hyperemia of the ears) were seen at a similar incidence in control animals. The porphyrin-like discharges noted in the 1000 and 2000 ppm males and females suggested that high concentrations of MIAK were somewhat irritating to the nose and eyes.

Subcutaneous masses (1.5 cm diameter) appeared on the ventral aspect of the necks of two 200 ppm males on days 25 and 28. For these animals, blood samples were taken at 4 week intervals and serum titers to sialodacryoadenitis virus were measured. The masses were consistent with the presence of salivary gland infections and did not appear to have any adverse effect on the animals or the study.

Body weights:
No significant effects on individual body weights or body weight changes were noted when MIAK-treated animals were compared to the controls. Mean terminal body weights for all animals were also comparable to corresponding controls.

Gross Pathology:
Gross examination revealed no MIAK-related changes.

Organ Weights:
Absolute and relative (to body weight) liver weights were increased relative to the control values in males and females at all concentrations in a dose-dependent manner. Statistical significance was only achieved at the two highest doses, 1000 and 2000 ppm. Absolute and relative kidney weights of males exposed to 1000 and 2000 ppm and relative kidney weights of females exposed to 2000 ppm were also statistically elevated. Although the mean absolute brain weights from females and relative heart weights from males exposed to 200 ppm were slightly statistically elevated relative to controls, these findings were considered spurious and not biologically significant. All other organ weights were comparable to controls.

A slight statistical increase in platelet counts in males exposed to 1000 and 2000 ppm was observed, but this increase was not considered biologically significant. Values for all other hematology parameters at all exposure levels were comparable to controls.

Serum clinical chemistries:
Values for all serum clinical chemistry parameters were comparable among treated and control groups.

In male rats, test substance-related histologic changes were noted in the liver and kidneys. In the liver, changes were characterized as minimal to moderate hypertrophy of hepatocytes (14/15; 2000 ppm), minimal to moderate eosinophilic cytoplasmic change (11/15; 2000 ppm), minimal to minor necrosis (10/15; 2000 ppm), minimal eosinophilic cytoplasmic change (5/15; 1000 ppm), minor hypertrophy of hepatocytes (5/15; 1000 ppm), and minimal necrosis (2/15; 1000 ppm). In the kidneys, the changes were characterized by minor to moderate regeneration of tubular epithelium (8/15; 2000 ppm and 7/15; 1000 ppm) and a possible increase in the severity of hyaline droplet degeneration in the proximal convoluted tubules. No compound-related changes were observed in males following exposure to 200 ppm MIAK. All other findings were either incidental in nature or occurred in a pattern similar to controls.

In female rats, test substance-related histologic changes were also noted in the liver and kidneys. In the liver, the changes were characterized as minimal to minor hypertrophy of hepatocytes (15/15; 2000 ppm and 15/15; 1000 ppm). In the kidneys the changes were characterized as minor tubular epithelium regeneration (6/15; 2000 ppm). No compound related changes were observed in females following exposure to 200 ppm MIAK. All other findings were either incidental in nature or occurred in a pattern similar to controls.

Effect levels

Dose descriptor:
Effect level:
200 other: ppm (target)
Basis for effect level:
other: Slight central nervous system effects, treatment-related organ weight changes, and histologic effects were observed in both male and female rats exposed to 1000 and 2000 ppm MIAK.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Chamber concentration:

The overall study mean exposure concentrations were 2079 ± 85, 1025 ± 29, 212 ± 14 and 0 ± 0 ppm compared to the target concentrations of 2000, 1000, 200 and 0 ppm, respectively. 

Nominal concentrations:

The overall study mean nominal concentrations were 1986 ± 29, 1011 ± 10, and 298 ± 35 ppm compared to the target concentrations of 2000, 1000, and 200 ppm, respectively. 

Chamber temperature:

The overall mean temperature for the 0, 200, 1000, and 2000 ppm groups was 22.9 ± 0.8°C, 22.8 ± 0.8°C, 22.8 ± 0.8°C and 22.8 ± 0.8°C, respectively. 

Chamber humidity:

The overall mean humidity for the 0, 200, 1000, and 2000 ppm groups was 44.9 ± 4.2%, 47.6 ± 4.6%, 39.2 ± 4.5%, and 42.9 ± 9.9%, respectively.

Applicant's summary and conclusion

When methyl isoamyl ketone was administered to rats via inhalation at concentrations of 0, 200, 1000, and 2000 ppm for 69 exposures spanning 96 days, the NOEC for systemic toxicity was determined to be 200 ppm based on treatment-related organ weight changes and histologic changes observed in the livers and kidneys of both males and females exposed to 1000 and 2000 ppm.

Although kidney weights were elevated in the 1000 and 2000 ppm exposure groups, there were no associated increases in urea nitrogen or creatinine. Hyaline droplet degeneration in the proximal convoluted tubular epithelium of the kidney is commonly seen in males of this strain of laboratory rat. Regeneration of tubular epithelium in both sexes was minor and neither kidney lesion impaired the health of the animals. Liver weight changes were accompanied by minor to moderate hepatocyte hypertrophy but no changes in serum clinical chemistries were observed.

Based on a clear NOEC of 200 ppm for repeated inhalation exposure 6 hr/day, 5 days/week for 13 weeks and the minimal to minor histopathologic effects observed at an exposure concentration of 1000 ppm, methyl isoamyl ketone is not classified for “Specific Target Organ Toxicity – Repeated Exposure” according to GHS guidelines.
Executive summary:

In a subchronic inhalation toxicity study, methyl isoamyl ketone (MIAK) was administered via inhalation to male and female rats at target concentrations of 0, 200, 1000, and 2000 ppm 6 hr/day for 69 exposures over 96 calendar days. No statistically significant or biologically relevant effects were noted in any group for body weight, hematology, serum clinical chemistry evaluations, or gross pathology. There was a dose-dependent statistically significant elevation of absolute and relative (to body weight) liver weights in males and females exposed to 1000 and 2000 ppm when compared to controls. Absolute and relative kidney weights were also elevated in males at the same two exposure concentrations, and relative kidney weights were elevated in females exposed to 2000 ppm. There were no increases in any serum clinical chemistries indicative of liver or kidney damage. Histopathological effects were observed in the liver and kidneys in both males and females exposed to MIAK at the two highest dose concentrations.  Effects were generally minimal to minor in both sexes, even at 1000 ppm. Under the conditions of this study, the NOEC in male and female rats exposed via inhalation for 13 weeks to methyl isoamyl ketone was 200 ppm.