Registration Dossier

Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1980
Report Date:
1980

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
-Test substance: Methyl isoamyl ketone (MIAK)
-Purity: 99.2%

Test animals

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.

Administration / exposure

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.
Doses / concentrations
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

Examinations

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%.

Results and discussion

Results of examinations

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.

Effect levels

Dose descriptor:
NOAEL
Effect level:
< 2 000 other: mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.