Registration Dossier

Toxicological information

Repeated dose toxicity: inhalation

Currently viewing:

Administrative data

Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Although the study was conducted prior to the introduction of GLPs, the study was conducted by the National Institute for Occupational Safety and Health using acceptable scientific methods. However, only a limited number of the parameters required for a subchronic or chronic inhalation guideline study were reported in the publication and only one sex was used.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1981

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
For the main study, groups of 50 male rats were randomly assigned to three treatment groups and were administered methyl n-amyl ketone at nominal exposure concentrations of 0 (control), 100, or 1000 ppm by whole-body inhalation 6 hr/day, 5 days/week for 10 months. A concurrent study conducted in monkeys is reported as a separate entry in this submission. The chronic rat study focused on several key areas: tissue pathology, metabolism, enzyme induction, and tissue distribution. Blood and urine were collected at study termination for metabolite identification. Liver microsomal enzyme induction was evaluated by assessing barbiturate-induced sleeping times. Tissue distribution analyses were conducted following both intraperitoneal (10 mg/kg) and inhalation exposure to [^14C]methyl n-amyl ketone. Tissues, urine and feces were collected 2, 4, 8, 12, 24, 48, and 72 hours after intraperitoneal administration of the radiolabeled test material. In the inhalation exposure studies, tissues were collected immediately or 4 hours after a 6-hr inhalation exposure to approximately 100 ppm of the labeled test material.
GLP compliance:
no
Remarks:
Conducted prior to GLPs
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Heptan-2-one
EC Number:
203-767-1
EC Name:
Heptan-2-one
Cas Number:
110-43-0
Molecular formula:
C7H14O
IUPAC Name:
heptan-2-one
Constituent 2
Reference substance name:
606-024-00-3
IUPAC Name:
606-024-00-3
Constituent 3
Reference substance name:
methyl amyl ketone; methyl pentyl ketone; MAK; 1-methylhexanal; butylacetone
IUPAC Name:
methyl amyl ketone; methyl pentyl ketone; MAK; 1-methylhexanal; butylacetone
Details on test material:
- Name of test material (as cited in study report): Methyl n-Amyl Ketone [Technical grade MAK]
- Source: Ashland Chemical Co., Division of Ashland Oil Co., Columbus, OH
- Physical state: liquid
- Analytical purity: 97%
- Impurities:

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
Separate groups of animals were used during various phases of this study.

TEST ANIMALS
- Source: Laboratory Supply Co., Inc., Indianapolis, IN
- Age at study initiation: approximately 2 months of age
- Weight at study initiation:
Animals in the original 10 month inhalation study: 258 ± 25g
Animals in the Tissue distribution (intraperitoneal) study : 229 ± 15g
Animals in the Tissue distribution (inhalation) study : 532 ± 52g (Body weights are significantly greater since this part of the study was performed approximately 6 months after the start of the 10-month inhalation study and animals were part of the original group allotted for the study.)
- Housing: Stainless- and galvanized-steel open-wire cages with wire mesh floors were used as housing. Rats were housed in groups of 2-4, one row above the other. Exposure group animals were housed in their chambers 24 hr/day; cage locations were rotated each month.
- Diet: Rodent Laboratory Chow, Ralston Purina Co., St. Louis, MO, ad libitum except during inhalation exposure
- Water: local municipality, ad libitum
- Acclimation period: 2 weeks prior to study initiation


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23-26
- Humidity (%): 40-50

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: room air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless Steel (6.37 m^3) inhalation chambers
- Method of holding animals in test chamber: Open wire cages with wire mesh floors were used to house animals to ensure adequate distribution of vapors during exposure. Rats were housed in groups of 2-4, one row above the other. Exposure group animals were housed in their chambers 24 hr/day; cage locations were rotated each month.
- Source and rate of air: For the 10-month study, inhalation exposures were carried out under dynamic flow conditions with tangential airfeed manifolds maintained at 40 L/min with a pressure of about 0.25 cm H2O.
- System of generating particulates/aerosols: Vapor concentrations of the test material were generated by heating liquid methyl n-amyl ketone in temperature-controlled flasks and using known air flows to carry the vapor into the exposure chambers.
- Temperature, humidity, pressure in air chamber: Temperature: 23-26 °C, Humidity: 40-50%


TEST ATMOSPHERE
- Brief description of analytical method used: Chamber test substance concentrations were monitored at least twice daily by pulling 10 and 5 liters of air from the 100 and 1000 ppm chambers, respectively, through charcoal tubes. Following desorption of the charcoal with carbon disulfide, the extract was analyzed by gas chromatography. Prior to the start of exposures, chamber concentrations were verified with gas chromatography. Chamber flow rates were adjusted as needed to maintain planned exposure levels. The average value for the daily samples was used to characterize methyl n-amyl ketone exposure on that day.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A Carle Model 8000 thermal conductivity gas chromatograph equipped with a stainless-steel 10% FFAP column was used for analyses of chamber concentrations during the exposure. A Perkin-Elmer Model 9000 gas chromatograph with flame ionization detector was used to analyze chamber concentrations prior to the start of exposure.
Duration of treatment / exposure:
A maximum of 10 months
Frequency of treatment:
6 hours/day, 5 days/week for main study
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
131 ± 30 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
1025 ± 136 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
50 male rats/group
Control animals:
yes, concurrent vehicle
Details on study design:
CONTROLS
- Animals were housed in separate animal facilities and were exposed to filtered air 24 hr/day.

METABOLISM STUDIES
- For animals exposed to unlabeled methyl n-amyl ketone for 10 months, blood was collected by cardiac puncture 1 hour after the final exposure. Urine was collected overnight (on ice) from fasted rats housed in individual metabolism cages. To detect high boiling metabolites, urine and serum samples were analyzed by gas chromatography using the method of DiVincenzo et al. (1976).

ENZYME INDUCTION STUDY
- The potential for methyl n-amyl ketone to induce liver microsomal enzymes was evaluated by comparing sleeping times following intraperitoneal injection of 25 mg/kg bw sodium pentobarbital into rats from each test and control group exposed for 10 months. Barbiturate administration took place about 18 hours after the end of the previous day’s methyl n-amyl ketone exposure.

TISSUE DISTRIBUTION STUDY (ip administration)
- [^14C]Methyl n-amyl ketone was diluted with unlabeled methyl n-amyl ketone and mixed with corn oil to give a concentration of 2.5 mg methyl n-amyl ketone/mL. Specific activity of the treatment solution was 4763 dpm/µg methyl n-amyl ketone. Twenty-one animals each received a dose of 10 mg methyl n-amyl ketone/kg bw (4 mL/kg bw). Tissues, urine and feces were collected from 3 rats per time interval at 2, 4, 8, 12, 24, 48 and 72 hours after dose administration. Sample preparation and counting were performed using the method of Plotnick and Conner (1976).

TISSUE DISTRIBUTION STUDY (inhalation exposure)
- Four groups of rats (3/group) were placed in individual glass metabolism chambers and exposed under dynamic conditions to radiolabeled methyl n-amyl ketone at approximately 100 ppm for 6 hours. Two of the groups contained animals from the 10-month inhalation study that had previously been exposed to unlabeled methyl n-amyl ketone at 131 ppm for 6 months prior to the tissue distribution study. The other two groups contained naïve animals with no prior exposure to methyl n-amyl ketone. One group of the previously exposed rats and one group of the naïve rats with no prior exposure to methyl n-amyl ketone were sacrificed immediately after the 6-hour exposure to [^14C]methyl n-amyl ketone. The remaining two groups of rats were sacrificed 4 hours later.

References:
DiVincenzo et al., 1976. Characterization of the metabolites of methyl n-butyl ketone, methyl iso-butyl ketone and methyl ethyl ketone in guinea pig serum and their clearance. Toxicol. Appl. Pharmacol., 36:511-522.
Plotnick HB and Conner WL, 1976. Tissue distribution of ^14C-labeled ethylene dibromide in the guinea pig. Res. Commun. Chem. Pathol. Pharmacol. 13:251-258.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Not reported

BODY WEIGHT: Yes
- Time schedule for examinations: Not reported

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

WATER CONSUMPTION: No data

Sacrifice and pathology:
GROSS PATHOLOGY:
- Animals were euthanized by a sodium pentobarbital (Nembutal Sodium, Abbott Laboratories, North Chicago, IL) overdose on the day following completion of inhalation exposure for 1, 3, 6 or 10 months. A gross necropsy was performed on all animals at each scheduled sacrifice.

HISTOLOGY:
- The following tissues were examined: lungs, heart, liver, spleen, kidneys, adrenals, pancreas, testes, and brain. The tibial branch of the sciatic nerve was also prepared for neuropathological examination.
Statistics:
Means and standard deviations were used as appropriate.

Results and discussion

Results of examinations

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 specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
- There were no clinical signs of toxicity. There was no test substance-related mortality during the study. Several rats died during the study but the deaths were attributed to chronic murine pneumonia.

BODY WEIGHT AND WEIGHT GAIN
- No effects

GROSS PATHOLOGY
- No effects were reported; all tissues were unremarkable.

HISTOPATHOLOGY: NON-NEOPLASTIC
- No tissue damage related to methyl n-amyl ketone exposure was observed.

OTHER FINDINGS
Liver microsomal enzyme induction:
-Based on an absence of significant differences in pentobarbital-induced sleeping times in rats exposed to concentrations of 0, 131 or 1025 ppm methyl n-amyl ketone for up to 10 months, the test chemical did not induce liver microsomal enzymes.

Tissue distribution studies:
-The mean air concentration of 132 ± 6.2 ppm methyl n-amyl ketone for the 6 hour inhalation exposures was similar to the lower exposure level used in the 10-month inhalation study. For both the intraperitoneal and inhalation routes of exposure, the highest level of radioactivity was found in the liver followed, in general, by the kidney, pancreas, and lung. Differences in tissue distribution of radioactivity did not correspond to any observed gross or histopathological damage since no target organ effects were observed at necropsy following a 10-month inhalation exposure and there were no significant differences between the intraperitoneal and inhalation routes of exposure. Because the structurally similar methyl n-butyl ketone has been linked to peripheral neuropathy in humans, potential target organs in this study were thought to be brain and sciatic nerves. For both ip and inhalation routes of exposure, very low levels of radioactivity were found in the brain and portions of sciatic nerves were below the limit of detection at all time intervals studied. The pattern of tissue distribution for rats exposed to radiolabeled methyl n-amyl ketone for 6 hours by the inhalation route was unaffected by prior exposure to unlabeled methyl n-amyl ketone for 6 months. Following intraperitoneal administration, most of the radioactivity derived from ^14C-methyl n-amyl ketone was recovered in the urine by 72 hours. Urinary excretion peaked at 12 hours (~25%) and was relatively constant at 24 and 48 hours for the three animals in each time group. Individual values at 72 hours accounted for 4.5, 13.7 and 33.3% of the administered dose and the differences in recovery could not be explained by water deprivation or lost urine volume. Less than 2% of the administered dose was recovered in the feces during that time period.

Metabolic studies:
-Methyl n-amyl ketone was detected and quantified in both serum and urine from rats exposed to both inhalation concentrations of methyl n-amyl ketone. Methyl n-amyl alcohol was not detected. While other metabolites were detected in both low and high exposure group serum and urine chromatograms in areas where the dione and the 2-keto-6-hydroxyheptane would be expected, these metabolites were not definitively identified and were reported only as “post methyl n-amyl ketone” peaks. No corresponding peaks were indentified in the urine or serum of control animals exposed to air only.

Effect levels

Dose descriptor:
NOEL
Effect level:
1 025 other: ppm (analytical) ± 136 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No effects were noted in any animal for any parameter examined during the study.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In a chronic inhalation study, groups of 50 male rats were exposed to methyl n-amyl ketone by whole body inhalation at exposure concentrations of 0, 131 ± 30 ppm or 1025 ± 136 ppm 6 hours/day, 5 days/week for 10 months. There were no statistically significant concentration-dependent effects on mortality, clinical signs or body weight gains. No gross or microscopic changes were reported in organs and tissues examined at necropsy. Analysis of blood and urine collected at study termination indicated the presence of parent compound as well as at least two unidentified metabolites at both exposure concentrations. Evaluation of potential liver microsomal induction by intraperitoneal injection of the barbiturate sodium pentobarbital into rats exposed by inhalation to 0, 131 or 1025 ppm methyl n-amyl ketone for 10 months indicated no significant differences in barbiturate-induced sleeping times.

Tissue distribution analyses conducted following a single intraperitoneal injection (10 mg/kg) or inhalation exposure to [14^C]methyl n-amyl ketone for 6 hours indicated that the administered radioactivity was rapidly eliminated in the urine with small amounts eliminated in the feces for both routes of exposure. Tissues collected 2, 4, 8, 12, 24, 48, and 72 hours after intraperitoneal administration of the radiolabeled test material and immediately or 4 hours after a 6-hour inhalation exposure indicated low levels of radioactivity in all tissues with highest levels in the liver, followed by kidney, pancreas and lung. There were lower levels in the brain and no detectable levels in the sciatic nerve. Based on the absence of clinical signs following long-term inhalation exposure to up to 1025 ppm and the absence of gross or microscopic changes observed at necropsy, methyl n-amyl ketone was not neurotoxic in rats under conditions of this study. The structurally similar chemical methyl n-butyl ketone has been linked to peripheral neuropathy in humans exposed to that chemical. Based on an absence of adverse effects in this study following long-term inhalation of methyl n-amyl ketone, the no-observed-effect-level (NOEL) was considered to be 1025 ppm in male rats.

The test material is not currently classified for target organ toxicity according to Annex I of Directive 67/548/EEC. Based on an absence of adverse effects observed in this study, methyl n-amyl ketone is not classified for Specific Target Organ Toxicity-Repeated Exposure according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 or the UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS).
Executive summary:

In a chronic toxicity study, groups of fifty male rats were exposed by whole-body inhalation to methyl n-amyl ketone at concentrations of 0, 131 ± 30 ppm or 1025 ± 136 ppm 6 hours/day, 5 days/week for 10 months.  There were no statistically significant concentration-dependent effects on mortality, clinical signs, or body weights and gross and microscopic examinations at study termination were normal. Parent compound as well as at least two unidentified metabolites were present in both serum and blood following long term exposure to either 131 or 1025 ppm methyl n-amyl ketone.  As evidenced by an absence of effects on barbiturate-induced sleeping times when compared to controls, long term inhalation of methyl n-amyl ketone did not induce rat liver microsomal enzymes. Tissue distribution studies comparing the inhalation and intraperitoneal routes of exposure indicated no significant differences from exposure to ^14C-methyl n-amyl ketone by either route and that prior long-term exposure to unlabeled methyl n-amyl ketone had no significant effect on tissue distribution of the radiolabeled material. Under conditions of this study, the no-observed-effect-level (NOEL) for methyl n-amyl ketone was determined to be 1025 ppm in male rats.