Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 weeks
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Although the study was conducted prior to the introduction of GLPs, the study was conducted by a credible testing facility, the British Industrial Biological Research Association, and the method used was similar to current OECD guidelines.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1972

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Fifteen animals/sex/group were administered methyl amyl ketone by oral gavage at dose levels of 0, 20, 100, or 500 mg/kg bw/day, 7 days/week for 13 weeks. Additional groups of 5 animals/sex, administered 0, 100, or 500 mg/kg bw/day of the test material, were terminated after 2 or 6 weeks of dosing. All animals were assessed for body weight, food and water intake, clinical chemistries, hematology, and urinalysis. After 13 weeks, all remaining animals were euthanized and a gross examination was performed with tissues saved for histopathology.
GLP compliance:
no
Remarks:
Study was 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 Amyl Ketone
- Physical state: Liquid
- Analytical purity: 98%
- Supplier: N.V. Chemische Fabriek "Naarden," Holland

Test animals

Species:
rat
Strain:
other: CFE
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: SPF breeding colony
- Age at study initiation: approximately 30 days old
- Weight at study initiation: males: 95-115g; female: 85-95g
- Housing: Animals were housed 5 per cage.
- Diet: Spillers' Laboratory Small Animal Diet (meal), ad libitum
- Water: local municipality, ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 1
- Humidity (%): 50-60


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- The test material was dissolved in corn oil at sufficient concentrations so that the dosing volume for all groups, including the control, was 5 mL/kg bw/day.

VEHICLE:
- A diet containing 1% methyl amyl ketone was prepared and placed in either a sealed container or allowed to be exposed to the air for 24 hours. Samples of each were then extracted and analyzed by gas chromatography. Eighty-five percent (85%) of the methyl amyl ketone was lost from the diet in 24 hours. Because of the high volatility of methyl amyl ketone and its insolubility in water, the test material could not be administered in the diet or drinking water and was therefore given daily per os in solutions of corn oil.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
20 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
0 mg/kg bw/day: 25 animals/sex
20 mg/kg bw/day: 15 animals/sex
100 mg/kg bw/day: 25 animals/sex
500 mg/kg bw/day: 25 animals/sex
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Behavior was assessed but no other details were provided.

BODY WEIGHT: Yes
- Time schedule for examinations: Initially; then weekly up to Week 12

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Food consumption was measured over the 24-hr period before the animals were weighed.

WATER CONSUMPTION : Yes
- Time schedule for examinations: Water consumption was measured over the 24-hr period before the animals were weighed.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Weeks 2, 6, and 13
- Anaesthetic used for blood collection: Yes, barbiturate anaesthesia
- Animals fasted: No data
- How many animals: Week 2: 5 animals/sex from control, 100 and 500 mg/kg bw/day groups; Week 6: 5 animals/sex from control, 100 and 500 mg/kg bw/day groups; Week 13: all remaining animals from all test and control groups
- Parameters examined: hemoglobin content, packed cell volume and counts of erythrocytes, reticulocytes, erythrocytes containing Heinz bodies, total leukocytes and the individual types of leukocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Weeks 2, 6, and 13
- Animals fasted: No data
- How many animals: Week 2: 5 animals/sex from control, 100 and 500 mg/kg bw/day groups; Week 6: 5 animals/sex from control, 100 (3 male and 5 female animals were examined) and 500 mg/kg bw/day groups; Week 13: all remaining animals from all test groups and control groups
- Parameters examined: Urea, glucose, total protein, and albumin; activities of glutamic-oxalacetic and glutamic-pyruvic transaminases and lactic dehydrogenase.

URINALYSIS: Yes
- Time schedule for collection of urine: Weeks 2, 6 and 13
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: During Week 2, urine was collected from 5 animals/sex in the control, mid- and high-dose groups and examined for appearance, number of cells excreted, and the presence of glucose, ketones, bile salts and blood. A concentration test was also performed to determine specific gravity and volume of urine produced during a 6-hr period of water deprivation. At Weeks 6 and 13, similar measurements to those mentioned above were conducted on the urine produced in a 2-hr period in 5 animals/sex in the control, mid- and high-dose groups following a water load of 25 mL/kg and that was produced between 16 and 20 hr after the water administration. At Week 6, the renal concentrating ability and urinary cell excretion were also determined in an additional six males from the control and 100 and 500 mg/kg bw/day groups. During Week 13, all tests were performed on 12 animals/sex in each dose group.

Sacrifice and pathology:
Gross pathology: All animals were euthanized with a barbiturate anesthesia followed by exsanguination of the aorta and examined for macroscopic abnormalities.

Organ weights: The brain, pituitary, thyroid, heart, liver, spleen, kidneys, adrenals, gonads, stomach, small intestine and caecum were weighed.

Histopathology: The brain, pituitary, thyroid, heart, liver, spleen, kidneys, adrenals, gonads, stomach, small intestine, caecum, salivary gland, trachea, aorta, thymus, lymph nodes, urinary bladder, colon, rectum, pancreas, uterus and skeletal muscle were preserved in 10% buffered formalin. Paraffin-wax sections of these tissues from the controls and high-dose group animals receiving the test material for 13 weeks were stained with haematoxylin and eosin for microscopic examination.
Statistics:
Data present in graphs and figures were compared using a Student’s t test.

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):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY
No effects on clinical signs. One female in the low dose group was euthanized during Week 12 because of a mass on the left side of the thorax. Histological examination showed the mass to be a mammary adenocarcinoma and no abnormalities were found in other tissues.

BODY WEIGHT AND WEIGHT GAIN
No effects. Body weights were comparable between the control and treated groups.

FOOD CONSUMPTION AND COMPOUND INTAKE
No effects. Food consumption values were comparable between the control and treated groups.

WATER CONSUMPTION AND COMPOUND INTAKE
No effects. Water consumption values were comparable between the control and treated groups.

HAEMATOLOGY
No effects. Haematology values were similar between the control and treated groups at all timepoints examined.

CLINICAL CHEMISTRY
No effects. Clinical chemistry values were similar between the control and treated groups at all timepoints examined.

URINALYSIS
In animals administered 100 or 500 mg/kg bw/day methyl amyl ketone for 13 weeks, there was a significantly increased number of cells excreted in the urine of both sexes compared to controls. A similar effect was observed in males after six weeks of treatment at the highest dose level tested.

ORGAN WEIGHTS
Absolute and relative liver weights were significantly higher than controls in both sexes of rats treated with 500 mg methyl amyl ketone/kg bw/day for 13 weeks, in 100 and 500 mg/kg bw/day dose-group males treated for 2 weeks, and in 500 mg/kg bw/day dose-group males treated for 6 weeks. In females, absolute but not relative liver weight was increased in the mid-dose group after 13 weeks. Statistically significant increases in kidney weights were observed in male rats only. Increases in both absolute and relative kidney weights were observed in male animals in the high-dose group at 13 weeks while absolute weight only was increased at 2 weeks. Relative kidney weight was increased in the mid-dose group after 13 weeks of treatment. Absolute and relative spleen weights of female rats dosed with methyl amyl ketone at 100 mg/kg bw/day for 6 weeks were significantly higher than controls but no effect was seen at higher dose levels, at other times points, or in male rats. Absolute but not relative stomach weight of female rats administered 500 mg/kg bw/day test material for 13 weeks was significantly higher than controls. A similar effect was not observed in male rats or in females at the other examination periods.

GROSS PATHOLOGY
No treatment-related dose-dependent abnormalities were observed at gross necropsy. No abnormalities were observed in either sex at Week 2. At Week 6, unilateral testicular atrophy was observed in a single male rat in the mid-dose group. No similar effect was observed in any other treatment group after more prolonged treatment. One low-dose group female was euthanized during Week 12 because of a large mass on the left side of the thorax. The mass was later identified as an adenocarcinoma. At Week 13, pale kidneys were observed in control and test animals.

HISTOPATHOLOGY: NON-NEOPLASTIC
The only histological finding observed was tubular dilation and atrophy in the kidneys of rats treated with 500 mg/kg bw/day of methyl amyl ketone for 13 weeks. The incidence and severity of this finding was similar in test and control rats.

HISTOPATHOLOGY: NEOPLASTIC
Prior to study termination, a mammary adenocarcinoma was identified in a single female rat in the lowest dose group. No other tumors were detected during the study at higher dose levels.

Effect levels

Dose descriptor:
NOEL
Effect level:
20 other: mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
When methyl amyl ketone was administered per os to male and female CFE rats daily for 13 weeks, the no-observed-effect-level (NOEL) was considered to be 20 mg/kg bw/day in male and female rats. There were no statistically significant dose-related adverse effects on mortality, appearance, behavior, body weight gains, food and water consumption, hematology, serum chemistries, or urinary concentration and dilution tests at any dose level tested. Although absolute and relative liver weights were increased in both sexes treated with 500 mg/kg bw/day of the test material for 13 weeks, there were no statistically significant changes in clinical chemistry parameters at any time period tested and no gross or histopathological changes were observed at necropsy. Statistically significant increases in kidney weights were limited to male rats only. While there was an increased excretion of cells in the urine of high-dose group male and female rats treated with methyl amyl ketone for 13 weeks and in high-dose group male rats treated for 6 weeks, tests for the presence of glucose, bile salts and blood were negative at all time periods, function in the concentration tests was unimpaired, and no significant changes were observed at histological examination. Tubular dilation and atrophy in the kidneys of high-dose group animals were similar in incidence and severity in treated and control rats.

The test material is not currently classified for target organ toxicity according to Annex I of Directive 67/548/EEC. Based on the limited effects observed in this study, methyl 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 subchronic toxicity study, fifteen to twenty-five rats/sex were administered methyl amyl ketone in corn oil by oral gavage at dose levels of 0, 20, 100, or 500 mg/kg bw/day for 13 weeks. No statistically significant effects were observed in mortality, clinical signs, body weights, food and water consumption, haematology, clinical chemistries, or urinary concentration and dilution tests at any dose level tested. Toxicity was limited to increased excretion of cells in the urine of male and female rats in the mid- and high-dose groups after 13 weeks and in male rats after 6 weeks and increases in absolute and/or relative weights of several organs in the absence of histopathological alterations. Under conditions of this study, the No-Observed-Effect-Level (NOEL) for daily exposure to methyl amyl ketone for 13 weeks was determined to be 20 mg/kg bw/day in male and female rats.