Butanone oxime

Neurotoxicity

Description of key information

Acute and subchronic exposure to high levels of MEKO resulted in transient neurotoxic effects

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

neurotoxicity: acute oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD guideline study conducted at reputable laboratory under GLP conditions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.6200 (Neurotoxicity Screening Battery)

GLP compliance:

yes

Remarks:

US laboratory, no certificate available

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 6 weeks old
- Housing: hanging-wire stainless steel cages
- Diet (e.g. ad libitum): Purina Certified Roden Chow (No. 5002), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2- 27.7
- Humidity (%): 47 +/- 15%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Frequency of treatment:

Single exposure

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

900 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes

Details on study design:

- Animals were followed for at least 14 days after dosing to observe for reversibility, persistence, or delayed occurrence
of toxic effects.
- A positive control group received 50 mg/kg acrylamide.
- A control group received distilled water.

Observations and clinical examinations performed and frequency:

Neuro behavioral assessments (functional observation battery and assessment of motor activity) were performed
before treatment, 1.6 and 22-26 hours following doing, and again 7 and 14 days after dosing.
- Additionally, clinical observations, periodic body weights and food consumption, and gross pathological examinations
were utilized to detect treatment-related effects.

Positive control:

Acrylamide

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:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Consistent treatment-related changes in neurobehavioral function were observed at 300 and 900 mg/kg/day. These changes were related to cage removal, handling, posture, gait, aerial righting, and motor activity following acute exposure. The nature of the effects suggests a transient narcoleptic response to high doses of MEKO characterized by motor in-coordination, decreased reactivity to general stimuli, and decreased motor activity. These effects last only a few hours. A single oral dose of MEKO to rats at a dose level of 100 mg/kg did not elicit any consistent or apparent treatment-related changes, therefore being the NOEL.

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

central nervous system

Organ:

brain

Treatment related:

yes

Dose response relationship:

yes

Conclusions:

Methyl ethyl ketoxime (MEKO) induced consistent treatment-related changes in neurobehavioral function of rats at oral doses of 300 and 900 mg/kg/day. A single oral dose of MEKO to rats at 100 mg/kg did not elicit any consistent or apparent treatment-related changes, therefore this dose level was considered the NOEL.

Reference 2

Endpoint:

neurotoxicity: sub-chronic oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OTS 798.6050 (Neurotoxicity Screening Battery)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 8 weeks
- Fasting period before study: no
- Housing: stainless steel with hanging-wire mesh cages
- Diet (e.g. ad libitum): Purina Certified Rodent Chow No. 5002, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1 - 27.7
- Humidity (%): 47 +/- 15%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

5 days/week

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Dose / conc.:

125 mg/kg bw/day (actual dose received)

Dose / conc.:

400 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Main study: 10/sex/dose
Satellite groups: 4/sex/dose assigned to control and high-dose recovery groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The 400 mg/kg bw/day dose of MEKO was chosen based on the results of prior repeateddose
toxicity studies and from results of acute and pilot neurotoxicity studies. The mid- and low-doses were spaced at
approximately one-half log units of the high-dose to ensure a dose-related gradient of effects and to obtain a NOEL.

Observations and clinical examinations performed and frequency:

- All animals were observed daily for mortality and for obvious indications of toxic effects.
- Detailed physical exams were conducted at each weighing interval. The body weights were recorded weekly.
- Food consumption was measured weekly.
- A Functional Observation Battery (FOB) and assessments of motor activity (MA) were performed prior to treatment
and 1,6 and 22-26 hours following the inital dose. Thereafter, behavioral assessments were conducted prior to
administration of test material on days 7, 14, 28, 56 and 91 to avoid measuring acute pharmacologic effects.
- After 13 weeks of exposure, animals were food-fasted overnight and blood samples were collected via orbital sinus
puncture under light anesthesiaHematology analysis was conducted using a S + IV Coulter Counter and
methemoglobin was determined using a IL282 Co-Oximeter. The followiing parameters were evaluated: hematocrit,
hemoglobin, erythroctye count, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration,
luekocyte count, leukocyte differential, platelet count, reticulocyte count, Heinz bodies, cell morphology and
methemoglobin.

Specific biochemical examinations:

- After 13 weeks of treatment, all rats were weighed and anesthetized by sodium pentobarbitol injection. Whole-body
perfusion was performed on the first 6 animals/sex/group using a gravity-fed infusion apparatus. Heparinized
physiological saline was perfused through the left ventricle, immediately followed by a buffered
glutaraldehyde/paraformaldehyde solution. All remaining animals were anesthetized and exsanguinated.
- Complete necropsies were performed on each animal. The brain, liver and spleen were trimmed and weighed from all
animals. Tissue samples from the central and peripheral nervous system were collected following whole-body in situ
perfusion and included the sciatic, tibial and sural nerves, lumbar and cervical dorsal and ventral roots, dorsal root
ganglion and gasserian ganglion, forebrain, midbrain, cortex, cerebellum, pons, medulla oblongata, and cervical (C3-
C6) and lumbar L1-L4) spinal cord
- Tissue samples from the high-dose MEKO animals, positive control animals and negative control animals were
trimmed and moistened with 0.1 M phosphate buffer (pH 7.4). The tissues were then processed through graded
alcohols and embedded in paraffin. Two sections (5 um) were cut from each sample and stained with either a Sevier-
Munger silver stain for visualizing axons or luxol fast blue with hematoxylin and eosin counter stain for visualizing
myelin and general cell morphology. Alls sections were examined by light microscopy.

Neurobehavioural examinations performed and frequency:

- A Functional Observation Battery (FOB) and assessments of motor activity (MA) were performed prior to treatment
and 1,6 and 22-26 hours following the inital dose. Thereafter, behavioral assessments were conducted prior to
administration of test material on days 7, 14, 28, 56 and 91 to avoid measuring acute pharmacologic effects.

Sacrifice and (histo)pathology:

The brain, liver and spleen were trimmed and weighed from all
animals. Tissue samples from the central and peripheral nervous system were collected following whole-body in situ
perfusion and included the sciatic, tibial and sural nerves, lumbar and cervical dorsal and ventral roots, dorsal root
ganglion and gasserian ganglion, forebrain, midbrain, cortex, cerebellum, pons, medulla oblongata, and cervical (C3-
C6) and lumbar L1-L4) spinal cord
- Tissue samples from the high-dose MEKO animals, positive control animals and negative control animals were
trimmed and moistened with 0.1 M phosphate buffer (pH 7.4). The tissues were then processed through graded
alcohols and embedded in paraffin. Two sections (5 um) were cut from each sample and stained with either a Sevier-
Munger silver stain for visualizing axons or luxol fast blue with hematoxylin and eosin counter stain for visualizing
myelin and general cell morphology. Alls sections were examined by light microscopy.

Positive control:

Acrylamide, dosed at 20 mg/kg bw/day; dose volume was 10 mL/kg bw. This dose was selected based on results in previous llterature reports and previous experience with acrylamide in the testing lab.

Statistics:

- Body weight, food consumption, clinical pathology, and organ weight data were analyzed using one-way analysis of
variance (ANOVA). When results from ANOVA were significant, group comparisons were made by Dunnett'smultiple/tests to determine significant differences form control.
- Behavioral parameters which yielded incremental (numerical) data were analyzed by using a two-way repeated
ANOVA. If significant treatment or treatment-time interactions were observed, then one-way ANOVAs were performed
at each time point. If significant treatment effects were observed at a given time point, then Dunnett's multiple t test was
conducted to determine significant differences from control. Data which were analyzed in this fashion included total (5-
minute) activity counts, rearing data, tail flick latencies, auditory startle data, grip strength data, number of fecal boli,
and number of urine pools.
- Behavioral parameters which yielded scaler or descriptive data suggestive of a treatment effect were analyzed by
using the repeated-measures SAS Catmod procedure. If significant treatment or treatment by time interactions were
evident, then univariate analysis was conducted at each time point.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Animals receiving 400 mg/kg bw/d were noted as having a pale appearance, hypoactivity, ataxia, excessive salivation, and dark colored urine at the post-dose cageside observations or during weekly physical examinations. These signs observed at cageside examinations generally were noticed shortly after dosing and were absent 24 hours post-dose. Of these signs, pale appearance and dark colored urine are probably related to the severe anemia which developed in the high-dose animals. The other signs of ataxia, and hypoactivity are consistent with an acute transient narcoleptic response to the high dose of MEKO and are consistent with the findings of the post-dose assessments and with a previous acute neurotoxicity study.

Mortality:

no mortality observed

Description (incidence):

Under the conditions of this study, oral administration of MEKO to rats 5days/week for 13 weeks at doses ranging from 40 to 400 mg/kg bw/day was not associated with significant mortality.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Under the conditions of this study, oral administration of MEKO to rats 5days/week for 13 weeks at doses ranging from 40 to 400 mg/kg bw/day was not associated with significant changes in 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:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The dose levels were associated with significant increases in methemoglobin values and hematological changes consistent with anemia.

Clinical biochemistry findings:

not specified

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Consistent treatment-related changes in neurobehavioural function were observed for cage removal, handling, posture, gait, arousal, salivation, approach response, rearing responses, and aerial righting for animals receiving 400 mg/kg bw/day. In general, these changes were transient occuring 1 hour after dosing and were not observed at pretreatment and during motor activity assessments (24 hours after dosing). No statistical differences were noted in the motoractivity data, although mean total activity counts in the high-dose animals were consistently lower than controls. No consistent changes in neurobehavioral function were noted in animals receiving 125 and 40 mg/kg bw/day.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The dose levels were associated with significant increased absolute and relative spleen weights.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The dose levels were associated with significant grossly enlarged spleens.

Neuropathological findings:

no effects observed

Description (incidence and severity):

Under the conditions of this study, oral administration of MEKO to rats 5days/week for 13 weeks at doses ranging from 40 to 400 mg/kg bw/day was not associated with significant microscopic changes in nervous tissue.

Dose descriptor:

NOAEL

Remarks:

neurotoxicity

Effect level:

125 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

Dose descriptor:

LOAEL

Effect level:

40 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

organ weights and organ / body weight ratios

Critical effects observed:

yes

Lowest effective dose / conc.:

400 mg/kg bw/day (actual dose received)

System:

central nervous system

Organ:

brain

Treatment related:

yes

Dose response relationship:

no

Critical effects observed:

yes

Lowest effective dose / conc.:

40 mg/kg bw/day (actual dose received)

System:

haematopoietic

Organ:

blood

Treatment related:

yes

Dose response relationship:

yes

Conclusions:

Oral administration of MEKO to rats at dose levels of 40 and 125 mg/kg/day for 13 weeks did not elicit any consistent or apparent treatment-related change in neurobehavioral function or nervous system structure while clearly producing changes in organ weight data and hematology parameters. Transient neurobehavioral changes occurred following MEKO doses of 400 mg/kg bw/day, were noted immediately after dosing, and had resolved by the next day. No progressive long term, irreversible neurotoxic changes were associated with MEKO administration as was seen with the positive control agent. Therefore, under the conditions of this study, 125 mg/kg bw/day was considered to be the no-observable-effect-level (NOEL) for neurobehavioral effects. Based on the hematological effects, the NOAEL for MEKO was considered < 40 mg/kg bw/day and LOAEL: = 40 mg/kg bw/day(hematological effects).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Species:

rat

Quality of whole database:

Both studies are performed according to the applicable guideline and under GLP, and have a Klimisch score of 1.

Effect on neurotoxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Effect on neurotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

 Following a single oral dose of the test substance to rats in an acute neurotoxicity study, transient effects characterized as motor incoordination were seen at treatment levels of 300 and 900 mg/kg bw/day but not at 100 mg/kg bw/day (IHF, 1993a). These effects included cage removal, handling, posture, gait, aerial righting, and motor activity following acute exposure. The nature of the effects suggests a transient narcoleptic response to high doses of the test substance characterised by motor in-coordination, decreased reactivity to general stimuli, and decreased motor activity. These effects last only a few hours. A single oral dose of the test substance to rats at a dose level of 100 mg/kg bw/day did not elicit any consistent or apparent treatment-related changes, therefore being the NOAEL.

In the 13-week neurotoxicity study in rats, rats were dosed 5 days/week by gavage for 13 weeks with 40, 125, or 400 mg/kg bw/day (IHF, 1993b). To check for neurotoxicity the standard "Functional Observational Battery", motor-activity data and neurohistopathology were performed. Acrylamide was used as a positive control. The test substance did not elicit any consistent or apparent treatment-related change in neurobehavioral function or nervous system structure. However, transient neurobehavioral changes (cage removal, handling, posture, gait, arousal, salivation, approach response, rearing responses, and aerial righting) occurred at doses of 400 mg/kg bw/day. They were noted immediately after dosing, and had resolved by the next day. No progressive long term, irreversible neurotoxic changes were associated with the test substance administration (as was seen with the positive control agent). Therefore, under the conditions of this study, 125 mg/kg bw/day was considered to be the NOAEL for neurobehavioral effects. For systemic toxicity a LOAEL of 40 mg/kg bw/day was derived based on haematological effects and changes in organ weights.

Justification for classification or non-classification

The available data on specific target organ toxicity - single exposure of the test substance meet the criteria for classification as STOT SE 3 (H336) according to Regulation (EC) 1272/2008.