4-methylpentan-2-one

Neurotoxicity

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Endpoint conclusion

Endpoint conclusion:

no study available

Effect on neurotoxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

neurotoxicity: sub-chronic inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

other: TSCA /FIFRA guidelines for neurotoxicity (US EPA Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals, addendum 10, Series 81, 82, and 83: EPA 540/09-91-123/ NTIS, PB-91-154617, 1991)

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Kingston, Stone Ridge, NY, USA
- Age at study initiation: 134 days (food restricted groups) or 68 days (ad libitum-fed groups)
- Weight at study initiation: 338 ± 12 g (food restricted groups) or 352 ± 12 g (ad libitum-fed groups)
- Fasting period before study:
- Housing: singly housed in stainless steel, wire-mesh cages during nonexposure periods
- Diet: PMI #5002, Purina Mills Inc, Richmond, IN, USA, ad libitum or food restricted to 14 grams/d
- Water: ad libitum
- Acclimation period: one-week

ENVIRONMENTAL CONDITIONS
No data

Route of administration:

inhalation: vapour

Vehicle:

clean air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 420 L stainless steel and glass New York University-type inhalation chambers (also known as Laskin chamber or Hinners chamber)
- Method of holding animals in test chamber: individual exposure cages. The location of each cage within the chamber was moved each week according to a defined rotation scheme.
- Source and rate of air:
- Method of conditioning air:
- System of generating vapors:The test atmosphere was generated by metering the MiBK liquid into glass distillation columns packed with glass beads. Filtered, compressed air was passed through the glass bead-packed columns to evaporate the solvent. The distillation columns were heated to ca. 50°C to enhance vaporization.
- Temperature, humidity:21.8-22.6°C, 54.6-66.8%
- Air change rate: 12 to 14 air changes per hour

TEST ATMOSPHERE
- Brief description of analytical method used: Chamber vapor concentrations were determined at least once each hour using a MIRAN IA infrared gas analyzer (Wilks Foxboro Analytical, South Nonvalk, CT) set at a wavelength of 8.6 µm.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours per day, 5 days per week

Remarks:

Doses / Concentrations:
250, 750 and 1500 ppm ( 1024, 3073, and 6146 mg/m3)
Basis:
other: target conc.

Remarks:

Doses / Concentrations:
255.7 ± 10.3, 754.9 ±12.0, 1519.4 ± 24.8 ppm
Basis:
analytical conc.

No. of animals per sex per dose:

20 (10 ad libitum-fed rats plus 10 food-restricted rats)

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: A concentration of 1500 ppm was selected as an exposure concentration that would produce overt signs of toxicity based on reduced activity during exposure and increased kidney weight in a two-week probe study. A concentration of 250 ppm was selected as an exposure concentration that was expected to have no effect. An exposure concentration of 750 ppm was selected as the intermediate exposure concentration.
- Rationale for animal assignment (if not random):
The population of ad libitum-fed animals were culled to a homogenous population based on body weight. The variation in body weight in the selected population did not exceed 20% of the mean. Animals from this selected population were then randomly distributed into treatment groups and the body weights for each group compared using an analysis of variance. The randomization procedure was accepted when no significant differences in body weight were seen among groups.
The animals for operant behavior testing were selected for randomization into groups based on their operant performance, and were placed into
treatment groups using a stratified randomization scheme. At the start of training, 80 male rats were fasted overnight. On the following day, the animals were placed into the operant chambers and provided a feed pellet once each minute following a 5 sec illumination of the cue lights (FR0). This continued until the animal touched the lever. The program then changed to a schedule of reinforce ment of each lever press with a feed pellet (FR1) until 30 pellets were received or 30 minutes elapsed in each component session. Following this first training step, the animals underwent six phases of training progressing from an FR1 schedule to a multiple FR10: 2FI60, followed by a FR20: 4FI60, 4FR20: 2FI60, 4FR20: FI120, and finally a 4FR20: 2FI120. Animals progressed from one training schedule to the next after they demonstrated proficiency by obtaining the maximum number of food pellets possible under a specific schedule. After 8 weeks, the population was culled to 60 animals based on performance in the multiple FR:FI schedule. Of the 60 animals, 40 were selected for study just prior to the initiation of exposures. Selection criteria included, but were not limited to coefficients of variation for FI response rate of 20%, index of curvature values of > 0.3, FI response rates of < 80 responses per min, and fixed ratio-to-fixed interval response rate ratio of > 1.5. Using this approach, "hand-shaping" was not necessary, and the performance indices of the population selected for the study were reasonably homogeneous. Using these selection criteria is not unlike selecting animals whose body weight falls within a narrow range for a study that measures body weight changes as an endpoint. Since the selection process occurred prior to exposure to the test substance, it did not influence the outcome of the study.
Animals that were culled in this procedure were humanely killed with carbon dioxide. The selected animail which were assigned to specific operant
chambers for the duration of the study. Exposures were initiated only after all the animals for the study demon­ strated stable behavior under the final multiple FR:FI schedule.
- Other: Exposures were initiated immediately after the last operant session (roughly 2 hours after the beginning of the light cycle).

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed once each hour during exposure. The outside wall of the chamber was scratched to assess the animals' alerh1ess. If the majority of animals in the group demonstrated similar signs, the observa tions were recorded for the entire group. The severity of response or behavior was graded as minimal, minor, mild, or severe. Before and after exposure, animals were handled and examined.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On weekends, cage-side observations were conducted. Observations included, but were not limited to, examination of the hair for discoloration and wctness, color of the skin, discharges from the eyes and mucous membranes, changes in motor activity, quantity and consistency of feces, discoloration of urine, changes in respiration, and changes in behavior patterns.

BODY WEIGHT: Yes
- Time schedule for examinations: For ad libitum-fed animals, body weights and food consumption were measured weekly prior to exposure. For SCOB animals, body weights were measured prior to each session.

FOOD CONSUMPTION:
Food consumption was not measured for SCOB rats because these animals typically consumed the entire daily allotment.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

Specific biochemical examinations:

Schedule-Controlled Operant Behavior (SCOB)
The equipment consisted of operant chambers (Model E10-09) in isolation cubides each containing a house light, three eue lights (over the lever), one lever (on the right sicle), a 2.5 kHz tone, and a food pellet dispenser. All equipment was purchased from Coulboum Instruments, Inc. (Allentown, PA). Chamber fonction was controlled by IBM-compatible personal computers (286 models; up to eight chambers per computer) using software provided by Coulbourn Instruments, Inc. Test sessions were conducted in a room separate from the housing room. Bedding material was placed in the trays under the SCOB chambers and was changed daily. Food pellet dispensers were cleaned weekly. SCOB chambers were cleaned weekly with an Akonox (Akonox, New York, NY) solution, after which the chambers were wiped down with 70% isopropyl akohol. The chambers were left vacant and open for at least 24 hours after cleaning.
The test animals' food was restricted to ca. 14 grams/day to motivate them; lever presses were reinforced with 45-mg food pellets {Dustless Precision
Pellets, BioServ Inc., Frenchtown, NJ, Cat. No. 0021, 45 mg). A multiple schedule of 4 fixed-ratio (FR) components (reinforcer after 20 responses; 4FR20) followed by 2 fixed-interval (FI) components (reinforcer after 120 sec; 2Fl120) was used. Lights above the lever served as the discriminating stimulus for the FR component, while a 2.5 kHz tone at 70 ± 2 dB served as the discriminating stimulus for the FI component. Each animal was assigned to a single test chamber at the beginning of the training period and was tested in this chamber throughout the study. The sessions were 47 minutes long and were conducted once a day, 5 days per week at the same time each day just prior to exposure. The sessions began within 30 minutes of the light cycle. The animals acquired the behavior over the course of several weeks using a weekly progression from continuous FR to a multiple FR10:FI60. The FR and FI
components were then increased to the final multiple FR:FI schedule. Stable behavior (coefficients of variation for index of curvature and FI of < 20% over 4 consecutive days) was demonstrated prior to the initiation of exposures. Animals were ca. 300 g at the start of the autoshaping. Operant behavior was measured for four consecutive days (Tuesday-Friday) prior to the first exposure to establish baseline response rates. Animals were divided into two replicates (twenty per replicate) evenly distributed across exposure groups and equipment. The parameters used to evaluate neurotoxicity were FR run rate, post-reinforcer pause duration, FI response rate, and index of curvature. The FR run rate is the number of lever presses per minute during the time interval from the first lever press to the 20th lever press. Lever presses that occurred during the first 250 msec were not counted (overflow responses). The postreinforcer pause duration is the time interval from the reinforcer of the last FR run to the next lever press. If overflow responses occurred during this time, the pause duration was the interval from the last overflow response to the first lever press of the next FR run. The rationale for disregarding overflow responses is that the post reinforcement pause may be misrepresented if overflow responses are used in the calculation. The FI response rate is the number of lever presses per minute during the time interval from the start of the FI run until the reinforcer. The index of curvature was cakulated using the method described by Fry et al. (1960) as adapted to FORTRAN by Maurissen and Inglis (1978). The index of curvature is the ratio of the area under the response-time curve within each of the 5 bins to the area under the hypothetical straight line of constant-rate responses. Weekly means of FR run rates, pause duration, FI response rates, and FI index of curvature values for each animal were compared with the individual animal baseline value, and the percent of baseline was calculated for each animal and group. Mean values for each parameter for each groups were also calculated. The data from Weeks 4, 8, and 13 were evaluated for evidence of neurotoxicity. These time intervals were selected to provide a snapshot of operant behavior performance during the course of the study, and to correspond to the time intervals recommended in the US EPA Neurotoxicity Screening Battery Testing Guideline for functional observational battery evaluations (FIFRA Series 81-8, 82-7, 83-1). Data were also collected for two weeks following the cessation of exposure.

Sacrifice and (histo)pathology:

All ad libitum-fed animals were fasted overnight prior to necropsy during Week 14, and the liver and kidneys of each animal were weighed. The liver and kidneys from five SCOB animals per group were also weighed at necropsy during Week 16. The remaining 5 SCOB animals per group were perfused through the ascending aorta with 4% paraformaldehyde followed by 5% glutaraldehyde, both in 0.1 M phosphate buffer, pH 7.4, at 4°C, and the brain, spinal chord, and peripheral nerves excised for possible neurohistopathology. No tissues were evaluated microscopically because there were no indications of neurotoxicity.

Statistics:

Body weight and food consumption data were evaluated using Bartlett's test (p <= 0.01) to test for equality of variances, one-way analysis of variance (ANOVA) (p <= 0.05), and Duncan's multiple range test (p = 0.05). SCOB data were analyzed as mean weekly percent of baseline values using ANOVA (p <= 0.05) and Dunnett's t-test (p <= 0.05), and time-concentration trends of the absolute values using a repeated measures analysis of variance (SAS Institute, Cary, NC). Operant behavior values at Week 13 and Week 15 were compared using a paired Student's t-test in order to evaluate the effect of cessation of exposure. A probability of p <= 0.05 (two­tailed) was used to determine significance.

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):

not examined

Ophthalmological findings:

not examined

Clinical biochemistry findings:

not examined

Behaviour (functional findings):

no effects observed

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
For Weeks 1 to 10, the animals exposed to 1500 ppm MiBK generally appeared normal for the first hour of the exposure, and then exhibited reduced activity of minimal to minor severity for the remainder of the day's exposure period. The 1500 ppm animals did not exhibit reduced activity at any time during the exposure period during Weeks 11 to 13 of the study. Reduced activity was defined as less movement, decreased alertness, and slower response to tapping on the chamber wall when compared with activity levels exhibited by control animals. Sialorrhea was observed in six 1500 ppm animals for no more than three exposure days. For Weeks 1 to 8, the animals exposed to 750 ppm generally appeared normal for the first two hours of the exposure, and then exhibited reduced activity of minimal severity for the remainder of the day's exposure period. The 750 ppm animals did not exhibit reduced activity at any time during the exposure period during Weeks 9 to 13 of the study. Sialorrhea was observed in three 750 ppm animals for no more than two exposure days. No effects on activity levels during exposure were observed in the 250 ppm group. In general, the clinical signs of the ad libitum-fed rats during exposure appeared similar to those of the SCOB animals. An exception was the occurrence of sialorrhea; the SCOB animais exhibited this clinical sign more frequently and in more animals than did the ad libitum-fed animals.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights for the ad libitum-fed groups exposed to MiBK were slightly higher than those observed for the control group; however, no statistical differences were noted. At the end of the in-life phase of the study, body weights for the ad libitum-fed groups exposed to MiBK were 5 to 7% higher than those for the control group.
Body weights for the 750 and 1500 ppm SCOB groups were also slightly higher than those for the control group. Although body weights were relatively stable at ca 340 g just prior to exposures based on a comparison of values at Day -10 and Day 0. By the end of the in-life phase, the mean body weights for the 750 and 1500 ppm groups were 5% higher than for the control group even though all the animals received only 14 g food/ day. Although the mean body weights were above the target of 300 g, no adjustments were made in the amount of food provided to the animals because this might have adversely affected SCOB performance.

FOOD CONSUMPTION AND COMPOUND INTAKE (IF FEEDING STUDY)
Mean food consumption for the ad libitum-fed groups exposed to MiBK was higher (p <= 0.05) than for the control group on Days 42, 49, 56, 70, 77, and 84.

NEUROBEHAVIOUR
The baseline performance of each group was comparable prior to exposure (Table 1), and no statistically significant differences in baseline performance were observed among groups at the beginning of exposure. No statistically significant differences in multiple FR:FI performance were noted among groups at any of the four specified time periods (Table 2). By Week 13, mean FR run rates increased 4-26%, in a non concentration dependent manner, and appeared to be unaffected by exposure to MiBK. Mean FR pause duration remained within 17% of baseline during Weeks 4, 8, and 13. Under the FI schedule, the mean response rate for the control group remained within 12% of baseline during Weeks 4, 8, and 13. Overall, the MiBK-exposed groups showed a gradual decrease in response rate throughout the study. Decreases in the mean response rate of > 10% were first observed at Week 8 for the 250 and 750 ppm groups, but not until Week 13 for the 1500 ppm group. By Week 13, the response rate for the MiBK-exposed groups were 22
to 25% below baseline rate. Mean index of curvature values remained within 18% of baseline during Weeks 4, 8, and 13. There were no statistically significant differences in operant parameters at Weeks 4, 8, or 13; there were no significant time-concentration interactions, and there was no significant difference between Week 13 (the last week of exposure) and Week 15 (two weeks following cessation of exposure) SCOB values.

GROSS PATHOLOGY
For SCOB animals, the mean tenninal body weights for the 750 and 1500 ppm groups were slightly higher than the mean weight for the control group, with the 1500 ppm group being significantly (p <= 0.05) higher (Table 3). The mean absolute liver weights for the 750 and 1500 ppm groups and the mean relative (to body weighl) liver weights for the 250 and 750 ppm groups were significantly higher (p <= 0.05) when compared to the mean weights of the control group. The mean absolute and relative (to body weight) kidney weights for the MiBK-exposed groups were comparable to those of the control group. No other organ weight differences were observed for SCOB anirnals.
For ad libitum-fed animals, the mean terminal.- body weights for the MiBK-exposed groups were comparable with those of the control group. The mean absolute liver and kidney weights for the MiBK-exposed groups and the relative (to body weight) liver and kidney weights for the 750 and 1500 ppm groups were significantly higher (p ::;; 0.05) when compared with the mean weights of the control group (Table 4). No other organ weight differences were observed.

Dose descriptor:

NOEC

Remarks:

neurotoxicity

Effect level:

>= 1 500 ppm

Sex:

male

Basis for effect level:

other: >= 6146 mg/m3

Remarks on result:

other:

Conclusions:

13 weeks of repeated exposure to concentrations of MiBK vapor that resulted in sedation during exposure did not significantly alter operant behaviour 16 hours after exposure as assessed by response rate, pause duration, or index of curvature. These data demonstrate the lack of persistent neurobehavioral effects and are consistent with reported results showing a rapid decline in blood levels of MiBK following exposure.

Executive summary:

The schedule-controlled operant behavior (SCOB) was evaluated to determine if subchronic exposure to MiBK vapor had the potential to alter behavior as an indicator of neurotoxicity. Food-restricted and ad libitum-fed Sprague-Dawley male rats were exposed to 0, 250, 750, or 1500 ppm MiBK for 6 h/day, 5 d/wk for 13 weeks. SCOB testing of food-restricted animals, using a multiple fixed ratio (FR)/fixed interval (FI) schedule (FR20:FI120), was conducted prior to each exposure to maintain the operant behavior; the data from Weeks -1, 4, 8, and 13 were evaluated for evidence of neurotoxicity. SCOB testing was also evaluated for two weeks following the cessation of exposures. Ad libitum-fed animals were included to assess systemic effects using routine indicators such as changes in body weight, food consumption, and organ weight. No significant differences were seen in fixed-ratio run rate, FR pause duration, fixed-interval response rate, and index of curvature values at any concentration. Animals exposed to 750 and 1500 ppm MiBK exhibited clinical signs associated with transient reduced activity levels, but only during exposure. No signs of reduced activity were observed immediately after exposure for either group. No other treatment-related abnormalities were observed during exposure. Food-restricted animals did not demonstrate any increased or decreased sensitivity to the CNS depressive effects of MiBK relative to the ad libitum-fed animals. No treatment-related body weight differences were observed within either the food-restricted groups or the ad libitum-fed groups, although body weights of the former were clearly depressed compared with those of the latter. Relative and absolute liver, and relative kidney weights were significantly greater for the 750 and 1500 ppm ad libitum-fed animals. No differences in kidney weight were observed for food-restricted animals, but absolute and/or relative liver weights were significantly higher for all the treated food-restricted groups. The results of this study indicate that repetitive exposures to high concentrations of MiBK vapors do not result in adverse effects on operant behavior in the rat.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

6 146 mg/m³

Study duration:

subchronic

Species:

rat

Effect on neurotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

David et al. (1999) exposed groups of 20 male Sprague-Dawley rats to 0, 250, 750, or 1500 ppm (0, 1024, 3073, and 6146 mg/m³) MIBK for 6 hrs/day, 5 days/week, for 13 weeks. One half of the rats at each exposure level was maintained on a restricted diet and the other half were fed ad libitum. All treatment groups were evaluated for changes in clinical signs, food consumption, body weight, organ weights (liver and kidney), and gross pathology (brain, spinal cord with dorsal and ventral roots, dorsal and ventral ganglia, sciatic nerve, tibial nerve, kidney, and liver); no histopathology was conducted. The treatment groups on restricted diets also underwent daily schedule-controlled operant behavioral (SCOB) testing from 4 days prior to exposure (to establish baseline responses), throughout the 13-week exposure period, and for 2 weeks following cessation of exposure. The SCOB testing in rats on the restricted diet consisted of fixed-ratio and fixed-interval schedules of reinforcement (food pellets) after appropriate response (lever press) to a cue (light or sound stimulus). Reduced activity during exposure was observed for the first 10 weeks (but not the final 3 weeks) of treatment in animals exposed to 6146 mg/m³and to a lesser degree in animals exposed to 3073 mg/m³during the first 8 weeks of treatment. No other treatment-related clinical signs were reported. Terminal mean body weights of restricted-diet rats were significantly greater than those of controls in groups exposed to 3073 or 6146 mg/m³. Terminal body weights of ad libitum-fed rats were 5–7% greater in exposed groups as compared to control groups, although no significant differences were observed. Mean relative liver weight was significantly higher in the 1024 and 3073 mg/m³groups (but not in the 6146 mg/m³group) among the restricted diet rats as compared to controls; among ad libitum-fed rats, both mean relative liver and kidney weights were significantly higher in the 3073 and 6146 mg/m³groups as compared to the control group. Food consumption was not consistently affected in rats fed ad libitum; food consumption was not measured in the rats fed restricted diets. Among restricted-diet rats, no significant differences between treatment groups and the control group were observed in any of the SCOB test measurements. No exposure-related gross pathologies were observed in either the restricted-diet or the ad libitum-fed treatment groups.

Justification for selection of effect on neurotoxicity via inhalation route endpoint:
Key study