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Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
90 days
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Although this study was conducted prior to GLPs and only one dose level was administered in the study, it is still a well-documented study that meets scientific principles.
Qualifier:
no guideline available
Principles of method if other than guideline:
The subchronic effects of methyl isoamyl ketone were investigated in male albino rats administered a maximum tolerated dose of 2000 mg/kg bw/day of the undiluted test substance per os 5 times a week over a 90 day period. Clinical observations, body weight gain, feed consumption, hematology, serum clinical chemistries, absolute and relative organ weights and gross and micropathology were recorded.
GLP compliance:
no
Remarks:
Study was conducted prior to GLPs
Limit test:
yes
Species:
rat
Strain:
other: CD COBS rats
Sex:
male
Details on test animals and environmental conditions:
Test animals:
-Strain: CD, COBS rats (Charles River Laboratories)
-Sex: Male
-Body weight: 287 ± 17g

Environmental conditions:
-Housing: individually in suspended wire-bottom cages fitted with galvanized pans to provide a smooth flooring.
-Bedding: Ab-Sorb-Dri®
-Feed: Purina Rodent Laboratory Chow 5001® ad libitum
-Water: Local municipality water ad libitum.
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
The subchronic effects of methyl isoamyl ketone were investigated in male albino rats administered a maximum tolerated dose of 2000 mg/kg bw/day of the undiluted test substance per os 5 times a week over a 90 day period. A concurrent control group was administered tap water at 4000 mg/kg bw/day following the same dosing regimen.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Gas chromatographic analysis of MIAK to determine the purity of the test substance was performed as part of a previously conducted acute neurologic study. Doses were based on animal body weights.
Duration of treatment / exposure:
90 days
Frequency of treatment:
5 days/week for 90 days.
Remarks:
Doses / Concentrations:
2000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
8 male rats
Control animals:
other: yes, control animals received 4000 mg/kg bw/day of tap water
Observations and examinations performed and frequency:
-Clinical signs: Animals were observed daily for signs of toxicity.

-Body weights: Animals were weighed twice weekly.

-Food consumption: Food consumption was determined twice weekly.

-Hematology: Prior to termination, blood was collected from the posterior vena cava for analysis of hematocrit, hemoglobin, white blood cell count and differential counts.

-Clinical chemistry: Prior to termination, blood was collected from the posterior vena cava for analysis of glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), lactic dehydrogenase, alkaline phosphatase, urea nitrogen and glucose.
Sacrifice and pathology:
-Animals found dead: Animals that died during the study were necropsied and a standard tissue set was collected, if possible.

-Scheduled necropsy: Animals that survived to study termination were euthanized and the following tissues were collected: trachea, lung, thymus, heart, tongue, esophagus, stomach, small intestine, large intestine, liver, kidneys, urinary bladder, adrenal glands, pancreas, thyroids, parathyroids, testis, epididymis, spleen, mensenteric lymph nodes, bone marrow, brain (medulla oblongata, cerebellum and cerebral cortex with thalamus and basal ganglia), spinal cord, sciatic-tibial nerves and dorsal root ganglia. Tissues were fixed in 10% buffered formalin, embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin-eosin. Quadriceps femoris, calf musculature and hind limb interosseous muscles were fixed in Bouin' s fixative. Eyes were fixed in a modified Zenker's fixative. Medulla oblongata, cerebellum and sciatic-tibial and plantar nerves were fixed in 5% glutaraldehyde in 0.lM phosphate buffer (pH 7.4 at 4°C); post-fixed in Dalton's chrome osmium solution, dehydrated in a series of ethanol and propylene oxide solutions, embedded in EPON 812, and sections were cut at 1 µm and stained with toluidine blue.

-Organ weights: Liver, kidney, brain, adrenal glands, testes, heart and spleen were weighed and relative organ weights calculated.

-Microscopic examination: All tissues from both the control and MIAK-exposed animals were examined by light microscopy.
Statistics:
Numerical data were analyzed by one-way analyses of variance (ANOVA), Bartlett's test and Duncan's multiple range test with statistical significance ascribed at 5%.
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):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
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:
-Mortality: No chemical-related mortality occurred.

-Clinical signs: No test substance related clinical abnormalities were found.

-Body weights: Mean body weights were statistically significantly lower than the controls during the first week of the study and from weeks 4 through the remainder of the study. Terminal body weights were statistically significantly lower than control body weights.

-Food consumption: There was a statistically significant reduction (23%) in feed consumption during the first week of the study. By week two, animals had adjusted to the treatment and there were no significant differences from the control during the remainder of the study.

-Hematology: There were no statistically significant effects on any hematology parameters when compared to controls.

-Clinical chemistries: There was a statistically significant increase in SGOT, SGPT and urea nitrogen but urea nitrogen levels were still within levels seen in historical controls.

-Organ weights: Absolute and relative liver weights, relative kidney weight, absolute and relative adrenal gland weights, relative brain weight and relative testes weight were significantly increased compared to controls. The increased relative testes and brain weights were attributed to lower body weight and all other organ weights were comparable to control.

-Gross necropsy: No gross pathological changes were observed.

-Microscopic examination: Microscopic changes were observed in the stomach, liver and kidneys. In the stomach, compound-related changes included hyperkeratosis, hyperkeratosis with pseudoepitheliomatous hyperplasia, and submucosal thickening and edema due to irritation following contact with the test material. Liver changes included diffuse hepatocyte hypertrophy and islands or nodules of strikingly different hepatocytes some of which were described as pre-neoplastic. Renal changes included an increased incidence of regenerating tubular epithelium, tubular dilation with casts and hyalin droplet formation in the proximal tubular epithelium.
Dose descriptor:
NOAEL
Effect level:
< 2 000 other: mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

Hepatic changes following MIAK exposure were different from that produced by other ketones and the control. In addition to diffuse hepatocyte hypertrophy which was seen in a number of structurally similar ketones and n-heptane, focal nodular changes were observed in four of seven surviving MIAK-exposed rats. These nodules were variable in appearance but all showed an increase in cyctoplasmic and, generally nuclear size. One type of nodule had a diffuse increase in cytoplasmic basophilia. The second type contained heavily vacuolated hepatocytes and the third had very large vesicular nuclei with very prominent nucleoli. All nodules were smaller than a hepatic lobule, but some compressed adjacent apparently normal, hepatic cords. Nodules of these types are generally regarded as pre-neoplastic changes. These changes are discussed in an attached memo from the attending pathologist, and show that the definition of "pre-neoplastic changes" used at the time the study was conducted are different than those used today (see attached memo in Section 13, Assessment reports, "MIAK Pathology Report").

Additional hepatic changes included necrosis of individual hepatocytes, vacuolation of individual hepatocytes, and bile duct epithelial hyperplasia.

Conclusions:
When methyl isoamyl ketone was administered to rats per os at a maximum tolerated dose level of 2000 mg/kg bw/day five days a week over 90 days, no NOAEL for systemic toxicity was determined. At the only dose-level tested, there was a statistically significant increase in SGOT and SGPT values and microscopic changes were observed in the stomach, liver and kidneys. Changes in the stomach were most likely due to irritation following contact with the test material and, as such, are not a true indication of a target organ effect. The observed kidney effects are consistent with development of chronic progressive nephropathy, a spontaneous lesion commonly found in male laboratory rats and which has not been reported in humans. Increases in certain liver enzymes, increases in absolute and relative liver weights, and histopathologic changes in the liver were all indicative of the liver as a target organ for exposure to MIAK.
Executive summary:

In a subchronic toxicity study, methyl isoamyl ketone (MIAK) was administered to male CD COBS rats by oral gavage at a maximum tolerated dose of 2000 mg/kg bw/day five days a week for 90 days. MIAK did not produce compound-related clinical abnormalities, deaths or gross pathological changes. Body weights and food consumption were decreased for the first week of the study; food consumption was comparable to control for the remainder of the study while body weights remained lower from weeks 4 through the remainder of the study. 

Hematologic determinations were all comparable to control values but certain clinical chemistry parameters including serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase were significantly increased compared to controls and correlated with observed hepatic lesions. Absolute and relative liver weights, relative kidney weight, absolute and relative adrenal gland weights, relative brain weight and relative testes weight were significantly increased compared to controls. There were no gross necropsy findings in animals that survived to study termination. 

Microscopic compound-related changes were found in the stomach, liver and kidneys. Changes in the stomach included hyperkeratosis, hyperkeratosis with pseudoepitheliomatous hyperplasia, and submucosal thickening and edema which were indicative of gastric irritation following contact with the test material. Liver changes consisted of a diffuse hepatocyte hypertrophy and microfoci of hyperplasia in some rats. The latter effect was characterized by an increase in cytoplasmic and, generally, nuclear size. Three different types of nodules were present. These types of nodules are generally regarded as pre-neoplastic changes.

These changes are discussed in an attached memo from the attending pathologist, and show that the definition of "pre-neoplastic changes" used at the time the study was conducted are different than those used today (see attached memo in Section 13, Assessment reports, "MIAK Pathology Report").Renal

changes included an increased incidence of regenerating tubular epithelium and dilatation with casts, and hyaline droplet formation in the proximal tubular epithelium. The observed renal changes are most likely associated with chronic progressive nephropathy, a condition commonly found in aging male laboratory rodents but which has no relevance for exposed humans.  

Under the conditions of this study, the NOAEL in male rats exposed via oral gavage to methyl isoamyl ketone subchronically was < 2000 mg/kg bw/day. 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
96 days
Reliability:
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
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
Deviations:
no
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:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
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
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
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:
yes
Details on analytical verification of doses or concentrations:
-Purity:
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
Remarks:
Doses / Concentrations:
200 ppm
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
1000 ppm
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
2000 ppm
Basis:
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.

Hematology:
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:
Euthanasia:
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.

Histopathology:
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.
Statistics:
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.
Clinical signs:
effects observed, treatment-related
Mortality:
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:
Mortality:
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.

Hematology:
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.

Histopathology:
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.
Dose descriptor:
NOEC
Effect level:
200 other: ppm (target)
Sex:
male/female
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.
Critical effects observed:
not specified

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.

Conclusions:
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.  

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
950 mg/m³
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

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

The potential for methyl isoamyl ketone to cause target organ toxicity following repeated exposure is well understood. Two guideline-equivalent 90-day repeat-exposure studies and two lesser 14-day subacute studies were available for review. In a study conducted by a method similar to OECD Guideline 413, male and female rats were exposed to up to 2000 ppm methyl isoamyl ketone via whole body inhalation for a total of 69 exposures over 96 days. There were no significant treatment-related effects on mortality, clinical signs, body weight and body weight gains, hematology, clinical chemistry parameters, or gross pathology. Target organ effects were limited to the liver and kidneys. Although there was a dose-dependent elevation in kidney weights in both sexes, there were no associated increases in urea nitrogen or creatinine indicating target organ damage. Histopathologic changes included a possible increase in hyaline droplet degeneration in the proximal convoluted tubular epithelium in male rats which is commonly seen in this strain of laboratory rat and minor regeneration of tubular epithelium in both sexes. 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. Severity of effects observed at 1000 ppm was minimal to moderate and there was a clear NOEC at 200 ppm for repeated inhalation exposure in rats exposed for 13 weeks.

Two lesser non-guideline subacute inhalation studies were also reviewed. In the first, in which a group of 5 rats (sex not reported) was exposed to 400 ppm of methyl isoamyl ketone for a total of 12 exposures over a 16 day period, there were no adverse effects on clinical signs, body weight gains, hematology, serum clinical chemistries, kidney and liver weights, or gross/microscopic liver or kidney pathology. The NOEC for this study was considered to be >400 ppm. In a second subacute inhalation study in which groups of male and female rats were exposed to up to 2000 ppm methyl isoamyl ketone for a total of 12 exposures over a 16-day period, the NOAEC was considered to be 1000 ppm based on the observation of slight central nervous system effects in both sexes at 2000 ppm and microscopic effects in the hearts of 2000 ppm females. Treatment-related hyaline droplet formation within cells of the proximal convoluted tubular epithelium in both male exposure groups was not considered an adverse effect since this effect is commonly seen in rats of this strain. It should be noted that cardiac effects were not observed in the 13-week study conducted at the same exposure concentration.

In a study conducted according to generally accepted scientific principles, 8 male rats were exposed to a maximum tolerated dose of 2000 mg/kg bw/day of methyl isoamyl ketone by oral gavage, five times per week over a 90-day period. There were no substance-related effects on mortality, clinical signs, hematology, or gross pathology. There were statistically significant increases in SGOT and SGPT values and microscopic changes were observed in the stomach, liver and kidneys. Compound-related changes in the stomach included hyperkeratosis, hyperkeratosis with pseudoepitheliomatous hyperplasia, and submucosal thickening and edema. Changes in the stomach were most likely due to irritation following contact with the test material and, as such, are not a true indication of a target organ effect. Renal changes included an increase in kidney weight which corresponded with an increased incidence of regenerating tubular epithelium, tubular dilation with casts and hyaline droplet formation in the proximal tubular epithelium. The observed kidney effects are consistent with the development of chronic progressive nephropathy, a spontaneous lesion commonly found in male laboratory rats and frequently exacerbated by chemical exposure. Similar kidney effects have not been reported in humans. The most significant effects occurred in the liver. Increases in absolute and relative liver weights along with two liver clinical chemistry parameters correlated with the observed hepatic lesions. Microscopic liver lesions included diffuse hepatocyte hypertrophy which has been seen following treatment with several structurally similar ketones. In addition, focal nodular changes were observed in four of seven treated animals. The nodules were variable in appearance but all showed an increase in cytoplasmic and, generally, nuclear size. Some nodules compressed adjacent apparently normal hepatic cords. Nodules of this type were described by the attending pathologist as suggestive of “pre-neoplastic” changes. These changes are discussed in an attached memo from the attending pathologist, and show that the definition of "pre-neoplastic changes" used at the time the study was conducted are different than those used today (see attached memo in section 13, assessment reports, "MIAK Pathology Report). Other hepatocyte changes included necrosis of individual hepatocytes, vacuolation of individual hepatocytes, and bile duct epithelial hyperplasia.

Following repeated oral exposures to a dose level of 2000 mg/kg bw/day, methyl isoamyl ketone caused focal hepatocyte hyperplasia in 4 of 7 rats comparable to those generally regarded as pre-neoplastic changes found in rodent species. However, only one high dose was tested (2000 mg/kg bw/day) and it was administered by a route not typical of industrial exposures. Since methyl isoamyl ketone has an appreciable volatility (VP = 4.5 mmHg at 25 °C), studies conducted by the inhalation route are probably more relevant to the evaluation of target organ effects following repeated exposure. When rats were exposed via inhalation for 90 days (at an exposure concentration comparable to the 2000 mg/kg bw/day oral dose), toxicity was not as extensive or severe and there were no signs of pre-neoplastic changes. Absorption and elimination studies conducted in rats by the oral and inhalation routes indicate there is a marked difference in the rate of blood clearance for the two routes of administration. Prolonged clearance following oral administration may explain the more significant liver toxicity observed in the 90-day oral study. 

To explore the effects of methyl isoamyl ketone on hepatic peroxisome proliferation, methyl isoamyl ketone was administered to rats by oral gavage at a dose of 2000 mg/kg bw/day for 14 doses over 19 calendar days. There is a body of data which links peroxisome-elicited hepatomegaly to the development of liver tumors in rats. Under conditions of this study, methyl isoamyl ketone was considered not to be an inducer of hepatic peroxisomes. Methyl isoamyl ketone did increase liver size (absolute and relative weight were increased), but there were no changes in hepatocyte size. Therefore, liver weight increases were related to increased numbers of hepatocytes rather than to hepatocyte hypertrophy.

Justification for classification or non-classification

When methyl isoamyl ketone was administered to rats via whole body inhalation at concentrations up to 2000 ppm for a total of 69 exposures spanning 96 days, the NOEC for systemic toxicity was determined to be 200 ppm based on treatment-related organ weight changes and microscopic effects observed in the livers and kidneys of both males and females exposed to 1000 and 2000 ppm. Increased kidney weights were not accompanied by changes in urea nitrogen or creatinine. The hyaline droplet degeneration in the proximal convoluted tubular epithelium of the kidneys seen in male rats is common for this strain of laboratory rat but has not been observed in humans. 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 which would be suggestive of liver damage. No hepatocyte hyperplasia was observed by the inhalation route. Based on a clear NOEC of 200 ppm following repeated inhalation exposure for 13 weeks and the minimal to minor nature of most effects observed even at 2000 ppm, methyl isoamyl ketone would not be considered a target organ toxicant by the inhalation route.

When methyl isoamyl ketone was administered to rats by oral gavage at a maximum tolerated dose level of 2000 mg/kg bw/day, five days/week over 90 days, there was a statistically significant increase in two liver enzyme values and microscopic changes were observed in the stomach, liver and kidney. Changes in the stomach were thought to be due to irritation following prolonged contact with the test material and, as such, would not be a true indication of a target organ effect. The observed kidney effects are consistent with those seen with exacerbation of a commonly occurring effect observed in male laboratory rats following chemical exposure and which have not been observed in humans. Increases in certain liver enzymes, increases in absolute and relative liver weights, and histopathologic changes in the liver were indicative of the liver as a target organ for exposure to methyl isoamyl ketone by the oral route. Although liver effects were observed at extremely high oral doses and only minor liver effects were observed at comparable inhalation exposures, in the absence of a NOEL or NOAEL by the oral route, methyl isoamyl ketone is considered to be a liver toxicant following repeated oral exposure.