5-methylhexan-2-one

Repeated dose toxicity: inhalation

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2 weeks

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Reliable with restriction; study was conducted in accordance with acceptable scientific standards; 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.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Rats were exposed to MIAK via inhalation 6 h/day, 5 days/week to target concentrations of 0, 1000, or 2000 ppm for 2 weeks (12 exposures). Clinical observations, body weights, hematology, and serum chemistry were collected on all animals. Animals were necropsied, and select organs were weighed and examined for any histopathological changes.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Strain: CRL:COBS®CD®(SD) BR
- Source of test animals: Charles River Breeding Laboratories, Wilmington, MA
- Weight at study start: males 217-259 g, females 139-199 g
- Quarantine period: 10 days
- Housing: Single 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:

- Animal 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.

- 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 (2.5 L MIAK in a 5.0 L bottle). To achieve concentrations of 2000 and 1000 ppm, the MIAK was heated to 47 or 32 °C, respectively. 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 / flow:
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 at least 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 valve. This system operated pneumatically.

-Chamber Temperature and Humidity:
Chamber temperature was recorded hourly. Chamber humidity was also recorded hourly with a modified Taylor hygrometer (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 2090 ± 77 and 803 ± 0 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:

12 total exposures in 16 days

Dose / conc.:

1 000 ppm

Remarks:

Basis: other: target concentration

Dose / conc.:

2 000 ppm

Remarks:

Basis: other: target concentration

No. of animals per sex per dose:

5 rats/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Thirty rats (15 male and 15 female) were divided into 6 groups of 5 rats each. Rats were exposed to target concentrations of 0, 1000, or 2000 ppm MIAK 6h/day, 5 days/week for a total of 12 exposures spanning 16 days. This exposure regimen was chosen in order to ensure at least 2 weeks of exposure.

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 twice 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 on Day 16 by exsanguination under sodium pentobarbital anesthesia.

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 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

Description (incidence and severity):

During inhalation exposure to 2000 ppm MIAK, both males and females exhibited slight lethargy and decreased aural response which were not observed with 1000 ppm MIAK. In MIAK-treated males the appearance of “gel-like” casts that had the appearance of a translucent rod 3-4 mm long and 1 mm in diameter were observed beneath the cages on day 14 only. The significance of this finding is unclear and may represent excretion of seminal fluid. All other clinical observations that occurred were seen with similar incidence in the control animals.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No effects; body weights and body weight gains of males and females exposed to MIAK were comparable to controls. Mean terminal body weights for all MIAK-treated animals were comparable to controls.

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

Description (incidence and severity):

Slight but statistical significant increases in polymorphonuclear leukocytes in the 2000 ppm male group and eosinophils in the 1000 ppm female groups were observed; neither of these changes were considered biologically significant. All other hematology parameters evaluated were comparable among groups.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

A slight decrease in alkaline phosphatase in the 2000 ppm male group and an increase in glucose in the 1000 ppm male group were observed, which were not considered biologically significant. All other serum clinical chemistry values were comparable among groups.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In all MIAK-treated animals, there was a slight increase in absolute and relative liver and kidney weights in both sexes in a dose-dependent manner. The increases reached statistical significance for relative liver and kidney weights in males and absolute and relative liver weights in females. The 1000 ppm relative kidney weights in females were also statistically increased. These findings were considered biologically significant. Absolute (2000 ppm) and relative (2000 and 1000 ppm) adrenal gland weights in MIAK treated females were statistically increased but were not considered of biological significance since there were no corresponding histopathological changes. All other organ weights were either comparable to controls or spurious and not considered biologically significant.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross examination revealed no MIAK-related changes at either exposure concentration.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

Clinical observations:
During inhalation exposure to 2000 ppm MIAK, both males and females exhibited slight lethargy and decreased aural response which were not observed with 1000 ppm MIAK. In MIAK-treated males the appearance of “gel-like” casts that had the appearance of a translucent rod 3-4 mm long and 1 mm in diameter were observed beneath the cages on day 14 only. The significance of this finding is unclear and may represent excretion of seminal fluid. All other clinical observations that occurred were seen with similar incidence in the control animals.

Body weights:
No effects; body weights and body weight gains of males and females exposed to MIAK were comparable to controls. Mean terminal body weights for all MIAK-treated animals were comparable to controls.

Organ weights:
In all MIAK-treated animals, there was a slight increase in absolute and relative liver and kidney weights in both sexes in a dose-dependent manner. The increases reached statistical significance for relative liver and kidney weights in males and absolute and relative liver weights in females. The 1000 ppm relative kidney weights in females were also statistically increased. These findings were considered biologically significant. Absolute (2000 ppm) and relative (2000 and 1000 ppm) adrenal gland weights in MIAK treated females were statistically increased but were not considered of biological significance since there were no corresponding histopathological changes. All other organ weights were either comparable to controls or spurious and not considered biologically significant.

Hematology:
Slight but statistical significant increases in polymorphonuclear leukocytes in the 2000 ppm male group and eosinophils in the 1000 ppm female groups were observed; neither of these changes were considered biologically significant. All other hematology parameters evaluated were comparable among groups.

Serum clinical chemistry:
A slight decrease in alkaline phosphatase in the 2000 ppm male group and an increase in glucose in the 1000 ppm male group were observed, which were not considered biologically significant. All other serum clinical chemistry values were comparable among groups.

Gross pathology:
Gross examination revealed no MIAK-related changes at either exposure concentration.

Dose descriptor:

NOAEC

Effect level:

1 000 other: ppm (target)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Histopathology:

A treatment-related effect in the kidneys of males was hyaline degeneration or hyaline droplet formation within cells of the proximal convoluted tubular epithelium and occasionally in the lumen of tubules. The significance of this histopathological lesion in this case is not clear. A second lesion that occurred in the heart of three 2000 ppm female rats was mineralization primarily involving the epicardium but to a lesser extent the myocardium. Some of the mineralized cells appeared hyperchromatic, but others appeared relatively normal. Even though this lesion appears to be related to MIAK exposure, it may be secondary to inflammatory lesions.

 

Chamber concentration:

The overall study mean exposure concentrations were 2086 ± 34, 974 ± 68, and 0 ± 0 ppm compared to the target concentrations of 2000, 1000, and 0 ppm, respectively. 

 

Nominal concentrations:

The overall study mean nominal concentrations were 2090 ± 77 and 803 ± 0 ppm compared to the target concentrations of 2000 and 1000, respectively. 

 

Chamber temperature:

The overall mean temperature for the 0, 1000, and 2000 ppm groups was 22.3 ± 0.3 °C, 22.2 ± 0.3 °C, and 22.0 ± 0.3 °C, respectively. 

 

Chamber humidity:

The overall mean humidity for the 0, 1000, and 2000 ppm groups was 44 ± 3 %, 35 ± 3 %, and 40 ± 5 %, respectively.  

Conclusions:

In a subacute toxicity study, methyl isoamyl ketone was administered via inhalation to 5 CRL:COBS® CD® (SD) BR rats/sex/dose at target concentrations of 0, 1000 and 2000 ppm for a total of 12 exposures in 16 days. A NOAEC for systemic toxicity of methyl isoamyl ketone was considered to be 1000 ppm based on slight central nervous system effects observed in both sexes at 2000 ppm and mineralization observed in the hearts of some 2000 ppm females. Histopathological kidney effects observed in male rats are commonly observed in laboratory rats of this strain, even in animals not subjected to chemical exposure. Based on the severity of the effects, this is not considered an adverse effect.

There were no significant treatment-related effects on body weight and body weight gain, hematology, clinical chemistry, or gross pathology in male and female rats exposed to a target concentration of 2000 ppm methyl isoamyl ketone for 2 weeks. Central nervous system effects were slight and observed only in the 2000 ppm group. Although there was a dose-related increase in liver weights in both sexes, there were no corresponding effects on liver enzyme levels or histopathology. Microscopic kidney effects were observed in male rats only and were of the type commonly seen in this strain of laboratory rat, even those without chemical exposure. Mineralization observed only in the hearts of some 2000 ppm females may be secondary to inflammatory lesions observed in this group. Based on the effects observed in this study, methyl isoamyl ketone is not classified for “Specific Target Organ Toxicity – Repeated Exposure” according to the EU-GHS guidelines.

Executive summary:

In a subacute toxicity study, methyl isoamyl ketone (MIAK) was administered via inhalation to groups of male and female CRL:COBS®CD®(SD) BR rats (5/sex/group) at target concentrations of 0, 1000 and 2000 ppm for 12 exposures over 16 days. No statistically significant or biologically relevant effects were noted in any groups for body weights, terminal body weights, hematology, serum clinical chemistry, or gross pathology. In all MIAK-treated animals, there was a slight, dose-dependent increase in absolute and relative liver weights in both sexes with no corresponding effects on serum clinical chemistries or histopathology. Histopathology revealed a treatment-related effect on hyaline droplet formation in the kidneys of male rats and mineralization involving the heart in 2000 ppm female rats. The kidney effects are commonly observed in this strain of laboratory rat, even those not subjected to chemical exposure. Mineralization in the heart may be related to inflammatory lesions in this group. Under the conditions of this study, the NOAEC following MIAK exposure for 2 weeks was considered to be 1000 ppm.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1983

Report date:

1983

Reference 2

Reference Type:

publication

Title:

Subchronic Inhalation Toxicity of Methyl isoamyl Ketone in Rats

Author:

Katz GV, Renner ER Jr. and Terhaar CJ

Year:

1986

Bibliographic source:

Fundamental and Applied Toxicology 6, 498-505

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

5-methyl-2-hexanone

IUPAC Name:

5-methyl-2-hexanone

Constituent 2

Reference substance name:

5-methylhexan-2-one

EC Number:

203-737-8

EC Name:

5-methylhexan-2-one

Cas Number:

110-12-3

Molecular formula:

C7H14O

IUPAC Name:

5-methylhexan-2-one

Constituent 3

Reference substance name:

MIAK; Isoamyl methyl ketone

IUPAC Name:

MIAK; Isoamyl methyl ketone

Details on test material:

-Test substance name (as cited in report): MIAK
-Source of test material: Tennessee Eastman Company, Kingsport, TN
-Appearance: clear

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Strain: CRL:COBS®CD®(SD) BR
- Source of test animals: Charles River Breeding Laboratories, Wilmington, MA
- Weight at study start: males 217-259 g, females 139-199 g
- 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

Vehicle:

other: Fresh air

Details on inhalation exposure:

- Animal 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 (2.5 L MIAK in a 5.0 L bottle). To achieve a concentration of 2000 ppm, the MIAK was heated to 47 °C. 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 / flow:
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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

200 ppm

Remarks:

Basis: other: target concentration

Doses / concentrations 2

Dose / conc.:

1 000 ppm

Remarks:

Basis: other: target concentration

Doses / concentrations 3

Dose / conc.:

2 000 ppm

Remarks:

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.

Examinations

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.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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.

Mortality:

mortality observed, treatment-related

Description (incidence):

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.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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.

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

Description (incidence and severity):

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.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

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

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

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.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross examination revealed no MIAK-related changes.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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.

Histopathological findings: neoplastic:

not examined

Other effects:

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.

Effect levels

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.

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

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.