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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted at a GLP facility under GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
MPK
Batch/Lot no. TD14042663
Exp. date: 11-Dec-2016
CAS no. 107-87-9
Clear, colorless liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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-exposure inhalation 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 used in this study (10/sex/group) was the minimum number of animals to allow for assessment of toxicity as required in the OPPTS 870.3465 and OECD 413 testing guidelines. Crl:CD(SD) rats (48 males and 48 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC, on 27-Jan-2015. The animals were approximately 37 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 with a subcutaneous microchip (BMDS system) implanted in the dorsoscapular area. All animals were housed for a 15-day acclimation period. During this period, each animal was observed twice daily for mortality and changes in general appearance or behavior. Data collection during acclimation began on 28-Jan-2015. 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 during acclimation.
All animals were housed 2 per cage by sex in clean, cages containing ground corncob bedding material (Bed O’Cobs®).. The cages were cleaned and changed routinely at a frequency consistent with maintaining good animal health. The bedding material is periodically analyzed by the manufacturer for contaminants. Analyses of the bedding material were provided by the manufacturer. No contaminants were present in the bedding at concentrations sufficient to interfere with the objectives of this study. The animals were temporarily separated as necessary to allow for the performance of protocol-specified activities. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011).
The basal diet used in this study, PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal), is a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research. Reverse osmosis-treated (on-site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the study, except during exposure periods or the scheduled period of fasting prior to necropsy when food, but not water, was withheld. Prior to the scheduled necropsy, food was offered for at least 2 hours after each animal’s final exposure prior to removal for the overnight fasting period. Municipal water supplying the facility was analyzed for contaminants according to SOPs. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study. 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 71 ± 5°F (22 ± 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. Actual mean daily temperature ranged from 70.0°F to 73.2°F (21.1°C to 22.9°C) and mean daily relative humidity ranged from 36.7% to 51.4% 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:
Animal exposures were conducted using four 1000-L glass and stainless steel whole-body inhalation exposure chambers. One exposure system was dedicated for each group for the duration of the study. A HEPA filter and an activated charcoal bed were used to pre-treat room air prior to delivery to the chambers. All test substance atmosphere chamber exhaust passed through the facility exhaust system, which included charcoal- and HEPA-filtration. The exposure period was 6 hours per day on a 5-day per week basis for 13 weeks (minimum of 65 exposures per animal). Animals were housed in a normal animal colony room during non-exposure hours. Food and water were withheld during each exposure period. For each day’s exposure, the animals were transferred to exposure caging in the colony room, transported to the exposure room, exposed for the requisite duration, and returned to their home cages in the animal colony room. The cage batteries were rotated on a daily basis between the 3 battery positions within the chambers to help ensure a similar exposure for all animals within each group over the duration of the exposure period.
The exposure chamber mean temperature and relative humidity were to be between 19°C to 25°C and 30% to 70%, respectively. All chambers were operated with at least 12 to 15 air changes per hour (200 to 250 standard liters per minute) and at a slight negative pressure. Oxygen content was measured during the method development phase and was 20.5% for Chambers 1, 2, and 3 and 20.4% for Chamber 4.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyzed exposure concentrations were determined at approximately 45-minute intervals using a gas chromatograph (GC). Samples were collected from the approximate animal-breathing zone of the exposure chamber via 1/4-inch polyethylene tubing. Under the control of the WINH system, sampling and analyses were performed as follows. An external multi-position valve 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 system then acquired the stored peak area data and used a natural logarithm quadratic equation based on the GC calibration curve to calculate the measured concentration in ppm.
Duration of treatment / exposure:
6 hours per day, 5 days per week for 13 weeks.
Frequency of treatment:
Daily, 5 days per week.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1500 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
750 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
250 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
0 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Methyl n-propyl ketone (MPK) was administered via whole-body inhalation exposure for 6 hours per day on a 5-day per week basis for 13 weeks (66 exposures for males and 67 exposures for females) to 3 groups of Crl:CD(SD) rats. Target exposure concentrations were 250, 750, and 1500 ppm.. A concurrent control group was exposed to humidified, filtered air on a comparable regimen. Mean analyzed exposure concentrations over the duration of the exposure period were 0, 251, 753 and 1499 ppm. Each group consisted of 10 animals/sex. Following 13 weeks of exposure, all animals were euthanized and necropsied. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed weekly (± 1 day). Arousal response observations were performed weekly during the exposure period. Individual body weights were recorded weekly (± 1 day). Cage food weights were recorded once weekly (± 2 days) beginning following randomization. Ophthalmic examinations were performed on study days -8 and 89. Clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were analyzed for all animals on the day of the scheduled necropsy (study week 13). Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsy. Selected tissues were examined microscopically from all animals.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
Clinical examinations were performed daily, prior to exposure and approximately 0-1 hour (+ 0.25 hour) following the end of exposure (designated as 1 hour post-exposure for report presentation purposes). On non-exposure days, 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 4 days of receipt, on the day of randomization, on the first day of exposure, weekly (± 1 day) during the study period, and on the day of the scheduled necropsy. Observations conducted prior to exposure were not performed on days when detailed physical examinations were conducted provided they were conducted prior to exposure. Observations on non-exposure days were not performed on days when detailed physical examinations were conducted. Due to social housing, some observations could not be attributed to a single animal. A separate computer protocol was used to record any observations noted outside of the above-specified intervals. 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-g 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 cotton 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).
Sacrifice and pathology:
Blood and urine samples for clinical pathology evaluations (hematology, coagulation,serum chemistry, and urinalysis) were collected from all animals on the day of the scheduled 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 retro-orbital sinus of animals anesthetized by inhalation of isoflurane. Blood was collected for coagulation parameters at the time of euthanasia via the vena cava of animals anesthetized by inhalation of isoflurane. Blood was collected into tubes containing K3EDTA (hematology), sodium citrate (coagulation), or no anticoagulant (serum chemistry).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed

Effect levels

Dose descriptor:
NOAEC
Effect level:
1 500 ppm
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Exposure of Crl:CD(SD) rats to MPK via whole-body inhalation exposure for 6 hours per
day on a 5-day per week basis for 13 weeks at exposure concentrations of 251, 753, and
1499 ppm was well tolerated. Test substance-related observations included increased
incidences of red material around the nose and on the facial area in all test
substance-treated group females and an increased number of ejaculatory plugs expelled
by the 1500 ppm group males during exposure. In addition, test substance-related
microscopic findings were limited to nasal level I, larynx, and thyroid in the males from
all exposure groups and/or 1500 ppm group females but were considered nonadverse.
Therefore, the no-observed-effect-concentration (NOEC) for males was not achieved and
the NOEC for females was 750 ppm. The no-observed-adverse-effect concentration
(NOAEC) was 1500 ppm for males and females, the highest concentration tested.
Executive summary:

The objective of this study was to evaluate potential toxic effects of methyl n-propyl ketone (MPK) when administered to Sprague Dawley rats via whole-body inhalation for 6 hours per day on a 5-day per week basis for 13 weeks (minimum of 65 exposures for each animal).

Methyl n-propyl ketone (MPK) was administered via whole-body inhalation exposure for 6 hours per day on a 5-day per week basis for 13 weeks (66 exposures for males and 67 exposures for females) to 3 groups (Groups 2-4) of Crl:CD(SD) rats. Target exposure concentrations were 250, 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, 251, 753 and 1499 ppm. Each group (Groups 1-4) consisted of 10 animals/sex. Following 13 weeks of exposure, all animals were euthanized and necropsied.

All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed weekly (± 1 day). Arousal response observations were performed weekly during the exposure period. Individual body weights were recorded weekly (± 1 day). Cage food weights were recorded once weekly (± 2 days) beginning following randomization. Ophthalmic examinations were performed on study days -8 and 89. Clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were analyzed for all animals on the day of the scheduled necropsy (study week 13). Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsy. Selected tissues were examined microscopically from all animals. There were no test substance-related effects on survival, arousal response, body weights, food consumption, hematology, coagulation, serum chemistry, and urinalysis parameters, or ophthalmic or macroscopic findings in any of the test substance-treated groups. A test substance-related increase in the incidence of red material around the nose and on the facial area was noted in females in all test substance-treated groups when compared to the control group at the 0-1 hour post-exposure observation session. Although similar observations were noted in males in all test substance-treated groups, the findings could not be attributed to the test substance as the incidence was similar across all groups including the control group. There was an increase in the number of ejaculatory plugs expelled by the 1500 ppm group males during exposure, generally beginning on study day 58 and continuing through the end of the exposure period. This increase was considered test substance-related. Test substance-related higher liver and thyroid/parathyroid weights were noted in the 1500 ppm group males. There were no test substance-related organ weight changes in the 250 and 750 ppm group males and females or the 1500 ppm group females. Test substance-related microscopic findings were limited to squamous epithelial hyperplasia in nasal level I of males in all test substance-exposed groups and 1500 ppm group females; higher incidence and severity of luminal exudate of the larynx in the 750 and 1500 ppm group males; and follicular cell hyperplasia of the thyroid gland in the 1500 ppm group males correlating with individual higher absolute and relative thyroid/parathyroid weight values.

Exposure of Crl:CD(SD) rats to MPK via whole-body inhalation exposure for 6 hours per day on a 5-day per week basis for 13 weeks at exposure concentrations of 251, 753, and 1499 ppm was well tolerated. Test substance-related observations included increased incidences of red material around the nose and on the facial area in all test substance-treated group females and an increased number of ejaculatory plugs expelled by the 1500 ppm group males during exposure. In addition, test substance-related microscopic findings were limited to nasal level I, larynx, and thyroid in the males from all exposure groups and/or 1500 ppm group females but were considered nonadvers. Therefore, the no-observed-effect-concentration (NOEC) for males was not achieved and the NOEC for females was 750 ppm. The no-observed-adverse-effect concentration (NOAEC) was 1500 ppm for males and females, the highest concentration tested.