3-methylbutanone

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 September 2016 to 17 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Regarding the inability to reference Annex XI. There was no potential for read-across to other registered substances (or ones with data). Since ketones do not have side groups that hydrolyze, the primary metabolic route is oxidation. While there is data for ketones with similar structures, they do not share common metabolites, and for those substances ECHA has already issued decisions stating that read-across to other ketones based on structural similarity is invalid. Since this is a 90-day study, there is no QSAR that can predict potential outcomes, especially in light of the requirement to do special staining for A2U in the kidney.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-methylbutanone

EC Number:

209-264-3

EC Name:

3-methylbutanone

Cas Number:

563-80-4

Molecular formula:

C5H10O

IUPAC Name:

3-methylbutan-2-one

Constituent 2

Reference substance name:

3-methyl-2-butanone

IUPAC Name:

3-methyl-2-butanone

Constituent 3

Reference substance name:

Methyl Isopropyl Ketone

IUPAC Name:

Methyl Isopropyl Ketone

Specific details on test material used for the study:

MIPK
Batch No. XK16013835
CAS No. 563-80-4
[ID No. 1602F9]
Clear, colorless liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Crl:CD(SD) rats were used as the test system for this study. This species and strain of animal is recognized as appropriate for repeated dose toxicity studies. The Sprague Dawley rat was selected because it is a widely used strain for which significant historical control data are available. The number of animals to be placed on study (10/sex/group) was based on the minimum number of animals required at the primary necropsy to be in accordance with the OPPTS 870.3465 Guideline. An additional 5/sex for the control and high-dose groups was included to evaluate the kidney following 6 weeks of exposure to MIPK. Crl:CD(SD) rats (58 males and 58 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC, on 20 Sep 2016. The animals were approximately 36 days old at receipt. Each animal was examined by a qualified technician on the day of receipt and weighed on the following day. Each animal was uniquely identified a subcutaneous microchip (BMDS system) implanted in the dorsoscapular area. All animals were housed for minimum of a 15-day acclimation. During acclimation, each animal was observed twice daily for mortality and changes in general appearance or behavior. Data collection during acclimation began on 21 Sep 2016. Individual body weights and cage food weights were recorded and detailed physical examinations were performed periodically during acclimation. Ophthalmic examination data were also recorded for animals prior to the initiation of dose administration.


All animals were housed throughout acclimation and during the study in an environmentally controlled room. The room temperature and relative humidity controls were set to maintain environmental conditions of 73°F ± 5°F (23°C ± 3°C) and 50% ± 20%, respectively. Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis. These data are summarized in Appendix 7. Actual mean daily temperature ranged from 72.2°F to 73.9°F (22.3°C to 23.3°C) and mean daily relative humidity ranged from 32.6% to 49.0% during the study. Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes. The 12-hour light/12-hour dark photoperiod was interrupted as necessary to allow for the performance of protocol-specified activities. Air handling units were set to provide a minimum of 10 fresh air changes per hour.

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

The exposures were conducted in 4 approximately 1000-L glass and stainless steel whole-body inhalation exposure chambers. One exposure chamber was dedicated to each group for the duration of the study. Animals were housed in a normal animal colony room during nonexposure hours. Prior to each exposure, the animals were transferred to exposure caging and transported to the exposure room. Animals were then exposed for the requisite duration and returned to their home cages. Animals were exposed while housed individually in standard exposure cage batteries of appropriate size for the whole-body chamber in use.
Air supplied to the whole-body chambers was provided from the Inhalation Department in-house nitrogen source and a HEPA- and charcoal-filtered, temperature and humidity-controlled supply air source. All chamber exhaust passed through the facility exhaust system, which consists of redundant exhaust blowers preceded by activated-charcoal and HEPA-filtration units.

Control Exposure Chamber (Group 1)
The control exposure chamber (Group 1, 0 ppm) was operated as follows. Humidified supply air was delivered to the exposure chamber from the Inhalation Department supply air source. To provide a comparable oxygen content to that of the treated groups, compressed nitrogen was delivered to the inlet of the exposure chamber, where it mixed with supply air. The amount of compressed nitrogen was controlled using a regulator and a flowmeter.


Test Substance Exposure Chambers (Groups 2–4)
A vapor atmosphere of MIPK was generated as follows. The MIPK vapors were generated using a separate glass-bead column-type vaporization system for each test substance exposure chamber. The column for each test substance exposure chamber was filled with various sized glass beads and heated using a heat tape controlled temperature controller and thermocouple. A FMI Lab Pump equipped with a piston head was used to deliver the liquid test substance from a reservoir to the top of each bead column. Nitrogen was metered to the bottom of each bead column using a regulator and a flowmeter. Vaporization occurred as the test substance flowed over the surface of the heated beads, while the nitrogen flowed up through the column. The concentrated MIPK vapors were directed through tubing through a 3-way bypass valve located prior to each exposure chamber inlet, where the concentrated vapors mixed with humidified supply air to achieve the desired target exposure concentration.

4.2.1. Nominal Exposure Concentrations
Nominal exposure concentrations were calculated for each test substance exposure chamber from the total amount of test substance used during each generation period and the total volume of air passed through the chamber during exposure. Test substance usage was determined by weighing the test substance container prior to and after the termination of generation. Total volume of air (including nitrogen from the vapor generation) was calculated by multiplying the daily mean ventilation rate by the duration of generation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyzed exposure concentrations were sampled and analyzed at approximately 45-minute intervals using gas chromatography (GC). Samples were collected from the approximate animal breathing zone of the exposure chamber via tubing. Under the control of the WINH system, sampling and analyses were performed as follows. The program controlled an external multi-position valve that permitted sequential sampling from the exposure room and each exposure chamber. Gas sampling injection onto the chromatography column occurred via an internal gas-sampling valve with a sample loop, the chromatograph was displayed and the area under the sample peak was calculated and stored. The WINH system then acquired the stored peak area data and used an ln-quadratic equation based on the GC calibration curve to calculate the measured concentration in ppm.

Duration of treatment / exposure:

Filtered air (control) and test substance atmospheres were administered as 6-hour, whole-body inhalation exposures on a 5-day per week basis to rats for 6 or 13 weeks (minimum of 30 or 65 total exposures). Five animals/sex in Group 1 and 4 were scheduled for an interim necropsy following at least 30 exposures. The remaining 10 animals/sex/group were scheduled for the primary necropsy after at least 65 exposures. On the day following each animal’s final exposure, all animals were euthanized and subjected to necropsy. The first day of dosing was Study Day 0; the first week of dosing was Study Week 0.

Frequency of treatment:

Filtered air (control) and test substance atmospheres were administered as 6-hour, whole-body inhalation exposures on a 5-day per week basis to rats for 6 or 13 weeks (minimum of 30 or 65 total exposures). Five animals/sex in Group 1 and 4 were scheduled for an interim necropsy following at least 30 exposures. The remaining 10 animals/sex/group were scheduled for the primary necropsy after at least 65 exposures. On the day following each animal’s final exposure, all animals were euthanized and subjected to necropsy. The first day of dosing was Study Day 0; the first week of dosing was Study Week 0.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Doses / concentrations 2

Dose / conc.:

300 ppm

Doses / concentrations 3

Dose / conc.:

750 ppm

Doses / concentrations 4

Dose / conc.:

1 500 ppm

No. of animals per sex per dose:

10 per sex per dose for the 90-day duration, 5 per sex at the control and high dose for the 30 day interem sacrifice.

Control animals:

yes, concurrent vehicle

Details on study design:

Methyl isopropyl ketone (MIPK; 3-Methylbutan-2one) was administered via whole-body inhalation exposure for 6 hours per day on a 5-day per week basis for 6 or 13 weeks (minimum of 30 or 65 total exposures) to 3 groups (Groups 2–4) of Crl:CD(SD) rats. Target exposure concentrations were 300, 750, and 1500 ppm for Groups 2, 3, and 4, respectively. A concurrent control group (Group 1) was exposed to humidified, filtered air on a comparable regimen. Mean analyzed exposure concentrations over the duration of the exposure period were 0, 304, 751, and 1508 ppm. Groups 1 and 4 each consisted of 15 animals/sex and Groups 2–3 each consisted of 10 animals/sex. Following 6 weeks of exposure, 5 animals/sex in Groups 1 and 4 were euthanized and necropsied; the remaining animals were euthanized and necropsied following 13 weeks of exposure.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

Survival
All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity
Clinical Observations
Clinical examinations were performed prior to exposure and 0–1 hour (+ 0.25 hr) following exposure (designated as 1 hour postexposure for report presentation purposes). On nonexposure days, the animals were observed once daily. The absence or presence of findings was recorded for individual animals at the scheduled intervals. Detailed physical examinations were conducted on all animals within 3 days of receipt, on the day of randomization, Study Day 0 (prior to exposure), weekly (± 2 days) during the study period, and on the days of the scheduled necropsies. Daily observations on nonexposure days were not necessary on days when detailed physical examinations were conducted provided they were conducted prior to exposure. In addition, the social groups were observed at the appropriate intervals for findings that could not be attributed to a single animal; only positive findings were recorded. A separate computer protocol was used to record any observations noted outside of the above-specified intervals and treatments. These unscheduled data are presented in Appendix 8.
Special attention was given to the state of arousal and response to novel stimuli during exposure (as close as possible to the end of the exposure period on a weekly basis) by producing a loud-noise stimulus. The noise was produced by allowing an approximately 50-gram item to strike the steel side of the exposure chamber at the approximate level of the cage rack. The stimulus item was attached to a length of rope that was held against the steel side of the chamber approximately 45 cm from the item. The stimulus item was raised until the rope was approximately perpendicular to the side of the chamber, and the item was released. The response to the stimulus was recorded for animals visible in the chamber as: not observed, no reaction, slight reaction (ear flick or some evidence that the stimulus was heard), or more energetic response (jump, flinch and/or vocalization)

Body Weights
Individual body weights were recorded within 3 days of receipt, on the day of randomization, Study Day 0 (prior to exposure), weekly (± 2 days) during the study period, on the day prior to the interim necropsy (nonfasted), and on the day prior to the first day of the primary necropsy (nonfasted). Mean body weights and mean body weight changes were calculated for the corresponding intervals. Final body weights (fasted) were recorded on the day of the scheduled necropsies.

Food Consumption
Cage food weights were recorded once weekly (± 2 days) beginning following randomization and throughout the study period. Food consumption was calculated as g/animal/day for the corresponding body weight intervals

FOB Assessments
FOB assessments were recorded for all remaining animals near the end of the exposure period (Study Week 11/12; designated at Study Week 12 on the data tables), following an exposure. The FOB utilized at Charles River is based on previously developed protocols. Testing was performed by the same biologists, whenever possible, without knowledge of the animal’s group assignment. The FOB was performed in a sound-attenuated room equipped with a white-noise generator set to operate at 70 ± 10 dB. All animals were observed for the following parameters as described below (refer to Appendix 9 for additional details on the FOB methodology and validation, and to Appendix 10 for a detailed description of the scoring criteria used for each observation):

Motor Activity
Motor activity was assessed for all remaining animals near the end of the exposure period (Study Week 11/12; designated at Study Week 12 on the data tables) following an exposure. Motor activity, recorded after the completion of the FOB, assessment was conducted using a personal computer-controlled system that utilizes a series of infrared photobeams surrounding an amber, plastic rectangular cage to quantify each animal’s motor activity. Four-sided black plastic enclosures were used to surround the transparent plastic boxes and decrease the potential for distraction from extraneous environmental stimuli or activity by biologists or adjacent animals. The black enclosures rested on top of the photobeam frame and did not interfere with the path of the beams. The motor activity assessment was performed in a sound-attenuated room equipped with a white-noise generator set to operate at 70 ± 10 dB. Each animal was tested separately. Data were collected in 5-minute epochs (print intervals) and the test session duration was 60 minutes. These data were compiled as six, 10-minute subintervals for tabulation. Data for ambulatory and total motor activity were tabulated. Total motor activity was defined as a combination of fine motor skills (i.e., grooming, interruption of 1 photobeam) and ambulatory motor activity (interruption of 2 or more consecutive photobeams).

Sacrifice and pathology:

Clinical Pathology
Blood and urine samples for clinical pathology evaluations (hematology, coagulation, serum chemistry, and urinalysis) were collected from all animals assigned to the interim (Study Week 6, urinalysis only) and primary necropsy (Study Week 13). The animals were fasted overnight prior to blood collection while in metabolism cages for urine collection. Blood was collected for hematology and serum chemistry evaluation via the jugular vein and for coagulation parameters at the time of euthanasia via the vena cava of animals anesthetized using isoflurane. Blood was collected into tubes containing potassium K2EDTA (hematology), sodium citrate (coagulation), or no anticoagulant (serum chemistry).

Hematology and Coagulation
Serum Chemistry
Urinalysis
Ophthalmic Examinations
Ocular examinations were conducted on all animals during acclimation (23 Sep 2016; Study Week -2) and near the end of the exposure period (03 Jan 2017; Study Week 12). All ocular examinations were conducted using an indirect ophthalmoscope and slit lamp biomicroscope preceded by pupillary dilation with an appropriate mydriatic agent.


Anatomic Pathology
Macroscopic Examination
A complete necropsy was conducted on all animals. Animals were anesthetized by isoflurane inhalation and euthanized by exsanguination. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. Clinical findings that were confirmed macroscopically were designated CEO on the individual macroscopic data tables. At the time of the interim necropsy, the kidneys were collected and placed in 10% neutral-buffered formalin. The left kidney was maintained in 10% neutral-buffered formalin for preparation of hematoxylin-eosin stained paraffin sections, while the right kidney was transferred to 70% ethanol after 48–72 hours for immunohistochemistry. The following tissues and organs were collected and placed in 10% neutral-buffered formalin for all animals at the primary necropsy

Organ Weights
The kidneys were weighed together from all animals at the interim necropsy and kidney to body weight ratio was calculated. The following organs were weighed from all animals at the primary necropsy:

Histology and Microscopic Examination
After fixation, protocol-specified tissues were trimmed according to Charles River SOPs and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned according to Charles River SOPs, mounted on glass microscope slides, and stained with hematoxylin and eosin. Histologic preparation of the hematoxylin-eosin stained paraffin sections were conducted at Charles River Ashland, and the preparation of the immunohistochemically stained sections were conducted at the Charles River site in Durham, NC (PAI Durham). Microscopic examination was performed on the kidneys and gross lesions at the interim necropsy. Hematoxylin-eosin stained paraffin sections were prepared of the left kidney (see Appendix 1 - Study Protocol and Deviations) and sections of the right kidney were immunohistochemically stained for α2μ globulin and histochemically stained with Mallory-Heidenhain to identify the presence of hyaline droplets. Microscopic examination was performed on all tissues listed in Section 5.9.1. from all animals in the control and 1500 ppm groups euthanized at the primary necropsy and on gross lesions and nasal cavity with turbinates (nasal levels 3, 4, and 5) from all animals in the 300 and 750 ppm groups euthanized at the primary necropsy. Additionally, slides from the left kidney were prepared from all males in the 300 and 750 ppm groups euthanized at the primary necropsy. Hematoxylin-eosin stained paraffin sections were prepared of the left kidney and sections of the right kidney were immunohistochemically stained for α2μ globulin and histochemically stained with Mallory-Heidenhain (a generic protein stain) to identify the presence of hyaline droplets. At least 1 representative image of a hematoxylin-eosin-stained kidney section and at least 1 representative image from a kidney section stained for α2μ globulin and with Mallory-Heidenhain were taken. Images were taken from control and 1500 ppm group males and females. Animals were selected at the discretion of the pathologist. Images were used for illustrative purposes only were not used for interpretation of pathological findings in the kidney. Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing, or other designations as appropriate. Tissues may appear on the report tables as not examined due to the tissue not being in the plane of section, not present at trimming, etc.

Statistics:

Statistical analysis is presented in the "any other information on materials and methods incl tables" section due to character limit restrictions in this entry field.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test substance-related clinical observations. There was an increased incidence of yellow material on various locations in the 750 ppm group males and 1500 ppm group males and females. This appeared to be a transient increase as it was only noted early in the study.

Test substance-related effects on arousal responses were observed in the 1500 ppm group males and females. No reaction to the arousal response stimulus was observed in the 1500 group males and females over the course of the study at an increased rate in comparison to the control group. This corresponded to a slight decrease in the incidence of and number of males and females with a slight reaction or more energetic response to the arousal response stimulus in the 1500 group animals in comparison to the control group. There were no adverse clinical observations related to hypoactivity, indicating that the decreased arousal at this dosage level did not likely persist following removal of the animals from the exposure chamber. Arousal response in the 300 and 750 ppm groups was unaffected by test substance exposure.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Test substance-related lower mean body weights (not statistically significant) and mean cumulative body weight gains (statistically significant through Study Week 5) were noted in the 750 and 1500 ppm group males throughout the course of the study. Test substance-related lower mean body weight gains were noted in the 750 and 1500 ppm group males and females during the first week of the study, but did not persist for the females. Although the differences in mean body weights did not reach statistical significance, they were considered test substance-related due to the differences noted in mean body weight gains. The differences in body weight and body weight gains in the 750 and 1500 ppm group males were considered nonadverse due to the magnitude of difference compared to concurrent controls.

There were no other test substance-related effects on body weight.

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:

no effects observed

Description (incidence and severity):

No ophthalmic lesions indicative of toxicity were observed in any of the test substance-exposed groups. Male No. 5520 and Female No. 5567 (1500 ppm group) were noted with unilateral retinal degeneration. This finding is recognized as a spontaneous occurring ocular abnormality and was not considered to be test substance-related because both eyes were not involved and larger areas of the retina were not affected. All findings observed were typical in prevalence and appearance for laboratory rats of this age and strain.

Haematological findings:

no effects observed

Description (incidence and severity):

Hematology and coagulation parameters were unaffected by test substance administration. Statistically significant higher mean white blood cell (WBC), absolute lymphocyte, and absolute monocyte counts were noted in the 1500 ppm group females; however, the values were within the Charles River Ashland (Version 3.6) historical control range. In addition, several individual control group animals had low values that contributed to a low mean value for the parameters.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum chemistry parameters were unaffected by test substance administration. Statistically significant lower mean glucose values were noted in the 1500 ppm group males at the primary necropsy. The difference was not considered test-substance because the individual values were minimally below the Charles River Ashland historical control range and the concurrent control group. Statistically significant lower mean albumin and total protein values were noted in the 750 and 1500 ppm group females at the primary necropsy. In addition, statistically significant lower mean calcium values were noted in the 300 and 1500 ppm group females at the primary necropsy. These differences were not considered test substance-related as the mean control values were on the high side of the Charles River Ashland historical control range, resulting in the statistically significant differences. Values from both the test substance-treated groups were also within the Charles River Ashland historical control range.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis parameters were unaffected by test substance administration. Urine pH in the 1500 ppm group males and females was lower in comparison to the control group; however, the values were within the Charles River Ashland historical control range.

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Test substance related organ weight changes were observed at the primary necropsy. The mean liver weight relative to final body weight was higher in the 1500 ppm group males and females and the 750 ppm group males. There were no other test substance-related changes.
At the interim necropsy, the kidney weight relative to final body weight was higher in the 1500 ppm group males. This finding was not observed at the primary necropsy. The magnitude of change was small and the values fell within the historical control reference range. The change was considered a result of individual animal variation rather than a test substance-related effect.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Test substance-related microscopic findings were noted in the olfactory epithelium of the nasal cavity of the 300, 750, and 1500 ppm group males and females. There was higher intensity of α2μ labeling in the kidneys of the 1500 ppm group males. Mallory-Heidenhain staining in the kidneys had a higher incidence in the 300, 750, and 1500 ppm group females compared to the 0 ppm group females, though all staining was minimal intensity with the exception of one 300 ppm female with mild intensity.
The histologic changes in the nasal cavity consisted of atrophy of the olfactory epithelium within nasal sections III, IV, and V of minimal to moderate severity. This was characterized by multifocal reduction in the number of layers of the olfactory epithelium (olfactory neurons and sustentacular cells) and vacuolation of the cytoplasm of some remaining cells, sometimes admixed with a little cellular debris and rare leukocytes. The lesions were most common in the dorsal medial meatus, but also affected smaller regions of the adjacent olfactory epithelium, such as the dorsal tip of the third turbinate and the nasal septum. There was a dose-response of the change from 300 to 750 ppm and less of a difference from 750 to 1500 ppm.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEC

Effect level:

1 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Highest level tested and no adverse effects found

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Executive summary:

The objective of the study was to evaluate the potential toxic effects of the test substance when administered via whole-body inhalation to Sprague Dawley rats for 6 hours per day on a 5-day per week basis for 6 or 13 weeks (minimum of 30 or 65 total exposures). Standard toxicity endpoints included clinical observations, body weights, food consumption, functional observational battery (FOB) and motor activity (MA) assessments, ophthalmic examinations, hematology and serum chemistry evaluations, urinalysis, macroscopic examinations and organ weight determinations at necropsy, and microscopic examinations. Additionally, microscopic evaluation was performed on kidney sections stained for Alpha-2μ (α2μ) and with the Mallory-Heidenhain (Mallory) stain.

The study was designed in general accordance with the US EPA Health Effects Test Guideline OPPTS 870.3465 (August 1998) and OECD Guideline for the Testing of Chemicals 413 (September 2009). Additionally, this study was conducted based on a REACh test order, which mandated immunohistochemical staining for α2μ as part of the study design. However, the signal for α2μ is known to diminish over time and approximately 28 days is considered to be the optimal exposure period, as compared to 90 days. Therefore, an interim necropsy subgroup was included for the control and high dose groups for animals exposed a minimum of 30 times. All groups were not included since establishing a dose response for α2μ staining was not the goal of the interim necropsy.

 

Methyl isopropyl ketone (MIPK; 3-Methylbutan-2one) was administered via whole-body inhalation exposure for 6 hours per day on a 5-day per week basis for 6 or 13 weeks (minimum of 30 or 65 total exposures) to 3 groups (Groups 2–4) of Crl:CD(SD) rats. Target exposure concentrations were 300, 750, and 1500 ppm for Groups 2, 3, and 4, respectively. A concurrent control group (Group 1) was exposed to humidified, filtered air on a comparable regimen. Mean analyzed exposure concentrations over the duration of the exposure period were 0, 304, 751, and 1508 ppm. Groups 1 and 4 each consisted of 15 animals/sex and Groups 2–3 each consisted of 10 animals/sex. Following 6 weeks of exposure, 5 animals/sex in Groups 1 and 4 were euthanized and necropsied; the remaining animals were euthanized and necropsied following 13 weeks of exposure.

 

All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed weekly (± 2 days). Arousal response observations were performed weekly during the exposure period. Individual body weights were recorded weekly (± 2 days). Cage food weights were recorded once weekly (± 2 days) beginning following randomization. Functional observational battery (FOB) and motor activity data were recorded for all animals during Study Week 11/12. Ophthalmic examinations were performed during Study Weeks -2 and 12. Clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were analyzed for all animals assigned to the interim (Study Week 6; urinalysis only) and primary (Study Week 13) necropsies. Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsies. Selected tissues were examined microscopically from all animals.

 

All animals survived to the scheduled necropsies. There were no test substance-related clinical observations, macroscopic findings, or effects on food consumption, functional observational battery assessments, motor activity, clinical pathology, or ophthalmic examinations.

 

Test substance-related effects on arousal responses were observed in the 1500 ppm group males and females. No reaction to the arousal response stimulus was observed in the 1500 group males and females over the course of the study at an increased rate in comparison to the control group. This corresponded to a slight decrease in the incidence of and number of males and females with a slight reaction or more energetic response to the arousal response stimulus in the 1500 group animals in comparison to the control group.

Test substance-related lower body weights and cumulative body weight gains were noted in the 750 and 1500 ppm group males throughout the course of the study. Test substance-related lower body weight gains were noted in the 750 and 1500 ppm group males and females during the first week of the study, but did not persist in females. The differences in body weight and body weight gains did not correlate with differences in food consumption.

Test substance-related higher liver weight to body weight ratio was observed in the 1500 ppm group males and females, as well as the 750 ppm group males. Test substance-related microscopic changes were observed in all test substance-treated groups, but were limited to minimal to moderate atrophy of the olfactory epithelium in the nasal cavity. A test substance-related slightly increased intensity in α2μ labeling of the kidney was observed in the 1500 ppm group males. However, there were no test substance-related effects noted during microscopic evaluation of the hematoxylin-eosin stained kidney sections. Based on the results of this study, exposure of MIPK to Crl:CD(SD) rats 6 hours per day on a 5-day per week basis for 6 or 13 weeks (minimum of 30 or 65 total exposures) at exposure levels of 300, 750, and 1500 ppm was well tolerated. Nonadverse test substance-related microscopic changes were observed in all test substance-treated groups, but were limited to minimal to moderate atrophy of the olfactory epithelium in the nasal cavity. A nonadverse, test substance-related increase in α2μ labeling of the kidney was observed in the 1500 ppm group males. Therefore, the no-observed-adverse-effect concentration (NOAEC) was considered to be 1500 ppm, the highest concentration tested on study.