Butanone oxime

Endpoint summary

Description of key information

Oral (equivalent to EPA OPPTS 870.3100), rat: LOAEL 25 mg/kg bw/day (hematological effects)

Inhalation (equivalent to OECD 413), mouse: NOAEC 10.8 mg/m³ (local effects)

Inhalation (equivalent to OECD 412), rat: NOAEC 90 mg/m³ (hematological effects)

A NOAEC of 54 mg/m³ was established for systemic effects in the 26-month carcinogenicity study with rats, based on changes in the liver.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Germantown, NY
- Age at study initiation: 6 weeks
- Housing: 5 animals per cage: polycarbonate (Lab Products, Inc., Rochelle Park, NJ), rotated twice per week; stainless steel racks
- Diet (e.g. ad libitum): NIH-07 Open Formula pelleted diet, Zeigler Brothers, Inc., Gardners, PA, available ad libitum, changed weekly
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14-17 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5-23.8
- Humidity (%): 50 +/-15%
- Air changes (per hr): at least 10 per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: drinking water

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulations and animal room samples were analyzed by gas chromatography. The analytical results for all dose formulations were within 10% of the theoretical concentrations.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily, ad libitum

Dose / conc.:

312 ppm

Remarks:

equivalent to 25 and 30 mg/kg bw/day (actual dose received) in males and females, respectively.

Dose / conc.:

625 ppm

Remarks:

equivalent to 50 and 65 mg/kg bw/day (actual dose received) in males and females, respectively.

Dose / conc.:

1 250 ppm

Remarks:

equivalent to 100 and 120 mg/kg bw/day (actual dose received) in males and females, respectively.

Dose / conc.:

2 500 ppm

Remarks:

equivalent to 175 and 215 mg/kg bw/day (actual dose received) in males and females, respectively.

Dose / conc.:

5 000 ppm

Remarks:

equivalent to 280 and 335 mg/kg bw/day (actual dose received) in males and females, respectively.

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

Observed twice daily for mortality/moribundity. Clinical findings were recorded weekly. Individual body weights were recorded at the start of the studies, weekly thereafter, and at the end of the studies. Water consumption was recorded twice per week and at the end of the studies.

Blood was collected from the retroorbital sinus of supplemental rats on days 5 and 21 and on all core study rats at study termination (week 13). Hematology: automated and manual hematocrit, hemoglobin concentration; erythrocyte, reticulocyte, and nucleated erythrocyte counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; platelet counts; leukocyte count and differentials; and methemoglobin.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At the end of the 13-week studies, necropsies were performed on all core-study animals. The heart, right kidney, liver, lungs, spleen, right testis, and thymus were weighed at necropsy.

HISTOPATHOLOGY: Yes
Histopathologic examinations were performed on all control and 5,000 ppm rats. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, brain, clitoral gland, esophagus, heart, large intestine (cecum, colon, and rectum), small intestine (duodenum, jejunum, and ileum), lung, lymph nodes (mandibular and mesenteric), mammary gland (with adjacent skin), ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, stomach (forestomach and glandular), right testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, and uterus. Organs examined at all exposure concentrations in rats were: bone with marrow, liver, nose, spl

Other examinations:

At the end of the 13-week studies, sperm samples were collected from all male rats in the 0, 1,250, 2,500, and 5,000 ppm groups for sperm motility evaluation. The following parameters were evaluated: spermatid heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and concentration. The left cauda epididymis, left epididymis, and left testis were weighed. Vaginal samples were collected for up to 12 consecutive days prior to the end of the studies from all female rats in the 0, 1,250, 2,500, and 5,000 ppm groups for vaginal cytology evaluations. The parameters evaluated were the relative frequency of estrous stages and estrous cycle length.

Statistics:

The Fisher exact test, a procedure based on the overall proportion of animals with specific lesions, was used to determine significance. Two approaches were employed to assess the significance of pairwise comparisons between exposed and control groups in the analysis of continuous variables. Organ and body weight data, which have approximately normal distributions, were analyzed with the parametric multiple comparisons procedures of Dunnett (1955) and Williams (1971, 1972). Hematology, spermatid, and epididymal spermatozoal data, which typically have skewed distributions, were analyzed with the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of exposure-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic exposure-related trend (Dunnett’s or Dunn’s test). Prior to statistical analysis, extreme values identified by the outlier test of Dixon and Massey (1951) were examined by NTP personnel, and implausible values were eliminated from the analysis. Because vaginal cytology data are proportions (the proportion of the observation period that an animal was in a given estrous stage), an arcsine transformation was used to bring the data into closer conformance with a normality assumption. Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across exposure concentrations.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The final mean body weights and body weight gains of the 2500 and 5000 ppm males were less than those of the controls as were the mean body weight gains of males in the 1250, 2500 and 5000 ppm groups and females in the 2500 and 5000 ppm groups.

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):

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology indicated a methemoglobinemia and a responsive Heinz body anaemia.

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Liver and spleen weights were generally significantly greater than those of the controls in the male and female rats exposed to 1250 ppm and higher. Kidney weights were significantly increased in males at 5000 ppm and females at 1250 ppm and above.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopically, there were exposure related increases in the incidences and severities of hematopoietic cell proliferation in the spleen at exposure concentrations of 625 ppm or greater in males and females. A significant increase in the incidence of hematopoietic proliferation in the bone marrow was observed in both sexes exposed to 625 ppm and greater. Liver Kupffer cell erythrophagocytosis and hemosiderin pigmentation, as well as renal tubule hemosiderin pigmentation, occured in exposed rats. Degeneration of the nasal olfactory epithelium occurred in male and female rats in the 2500 and 5000 ppm groups.

Histopathological findings: neoplastic:

no effects observed

Details on results:

A NOAEL for the most sensitive parameter (erythrotoxicity) for rats was 312 ppm.

Dose descriptor:

NOAEL

Remarks:

erythrotoxicity

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: erythrotoxicity

Dose descriptor:

NOAEL

Remarks:

erythrotoxicity

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: erythrotoxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

haematopoietic

Organ:

blood

Treatment related:

yes

Dose response relationship:

yes

Conclusions:

Based on results of this 13-week oral toxicity study of methyl ethyl ketoxime (MEKO) in rats, the major target organ of MEKO is the erythrocyte. The NOAEL is 625 ppm in male rats and 312 ppm in female rats based on erythrocyte counts. The NOAEL for hematopoietic toxicity is 312 ppm for both sexes based on erythroid cell hypeerplasia in bone marrow. Hematology indicates that MEKO induces a methemoglobinemia and a responsive Heinz body anemia.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13-weeks

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: EPA 798.6050, 798.6200, 798.6400,

Principles of method if other than guideline:

To evaluate toxicity including neurotoxicity following 90 days of exposure.

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 oral 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

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 repeated-dose 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.

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.

Observations and 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.

Sacrifice and pathology:

- 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.

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's multiple/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 postdose cageside observations or during weekly physical examinations. These signs observed at cageside examinations generally were noticed shortly after dosing and were absent 24 hours postdose. 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 postdose assessments and with a previous acute neurotoxicity study. No consistent changes in neurobehavioural function were noted in animals receiving 125 and 40 mg/kg bw/day.

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 specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

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

Clinical biochemistry findings:

not specified

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

At 400 mg/kg bw/day, dark colored urine was observed.

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 motor-activity data, although mean total activity counts in the high-dose animals were consistently lower than controls.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

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

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The dose levels were associated with grossly enlarged spleens.

Neuropathological findings:

no effects observed

Description (incidence and severity):

No microscopic changes observed in nervous tissue at doses ranging from 40 to 400 mg/kg bw/day.

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not examined

Dose descriptor:

NOAEL

Remarks:

neurobehavioral effects

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: transient neurobehavioral changes

Dose descriptor:

LOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: haematological effects and changes in organ weights

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.
LOAEL: = 40 mg/kg bw/day(hematological effects)

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Germantown, NY
- Age at study initiation: 6 weeks
- Housing: 1 animal per cage; polycarbonate (Lab Products, Inc., Rochelle Park, NJ) rotated once per week; stainless steel racks
- Diet (e.g. ad libitum): NIH-07 Open Formula pelleted diet, Zeigler Brothers, Inc., Gardners, PA, available ad libitum, changed weekly
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5-23.8
- Humidity (%): 50 +/-15%
- Air changes (per hr): at least 10 per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: drinking water

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulations and animal room samples were analyzed by gas chromatography. The analytical results for all dose formulations were within 10% of the theoretical concentrations.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily, ad libitum

Dose / conc.:

625 ppm

Remarks:

equivalent to 110 and 145 mg/kg bw/day in males and females, respectively.

Dose / conc.:

1 250 ppm

Remarks:

equivalent to 200 and 340 mg/kg bw/day in males and females, respectively.

Dose / conc.:

2 500 ppm

Remarks:

equivalent to 515 and 630 mg/kg bw/day in males and females, respectively.

Dose / conc.:

5 000 ppm

Remarks:

equivalent to 755 and 1010 mg/kg bw/day in males and females, respectively.

Dose / conc.:

10 000 ppm

Remarks:

equivalent to 1330 and 3170 mg/kg bw/day in males and females, respectively.

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

Observed twice daily for mortality/moribundity. Clinical findings were recorded weekly. Individual body weights were recorded at the start of the studies, weekly thereafter, and at the end of the studies. Water consumption was recorded twice per week and at the end of the studies.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At the end of the 13-week studies, necropsies were performed on all core-study animals. The heart, right kidney, liver, lungs, spleen, right testis, and thymus were weighed at necropsy.

HISTOPATHOLOGY: Yes
Histopathologic examinations were performed on all control and 10,000 ppm mice. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, brain, clitoral gland, esophagus, gallbladder, heart, large intestine (cecum, colon, and rectum), small intestine (duodenum, jejunum, and ileum), lung, lymph nodes (mandibular and mesenteric), mammary gland (with adjacent skin), ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, stomach (forestomach and glandular), right testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, and uterus. Organs examined at all exposure concentrations in female mice were: bone with marrow, liver, nose, spleen, and urinary bladder. The kidney was examined in 0, 625, 5,000 and 10,000 ppm male mice, and in 0 and 10,000 ppm female mice. Organs examined in 0, 625, 2,500, 5,000, and 10,000 ppm male mice were: liver, nose, and spleen. The bone with marrow was examined in 0, 625, 5,000, and 10,000 ppm male mice.

Other examinations:

At the end of the 13-week studies, sperm samples were collected from all male mice in the 0, 2,500, 5,000 and 10,000 ppm groups for sperm motility evaluation. The following parameters were evaluated: spermatid heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and concentration. The left cauda epididymis, left epididymis, and left testis were weighed. Vaginal samples were collected for up to 12 consecutive days prior to the end of the studies from all female mice in the 0, 2,500, 5,000 and 10,000 ppm groups for vaginal cytology evaluations. The parameters evaluated were the relative frequency of estrous stages and estrous cycle length.

Statistics:

The Fisher exact test, a procedure based on the overall proportion of animals with specific lesions, was used to determine significance. Two approaches were employed to assess the significance of pairwise comparisons between exposed and control groups in the analysis of continuous variables. Organ and body weight data, which have approximately normal distributions, were analyzed with the parametric multiple comparisons procedures of Dunnett (1955) and Williams (1971, 1972). Hematology, spermatid, and epididymal spermatozoal data, which typically have skewed distributions, were analyzed with the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of exposure-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic exposure-related trend (Dunnett’s or Dunn’s test). Prior to statistical analysis, extreme values identified by the outlier test of Dixon and Massey (1951) were examined by NTP personnel, and implausible values were eliminated from the analysis. Because vaginal cytology data are proportions (the proportion of the observation period that an animal was in a given estrous stage), an arcsine transformation was used to bring the data into closer conformance with a normality assumption. Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across exposure concentrations.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights and body weights gains of the 10000 ppm males and females were less than those of the controls.

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):

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology indicated a methemoglobinemia and a responsive Heinz body anaemia.

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Spleen weights were generally significantly greater than those of the controls in the male and female mice exposed to 10000 ppm.

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopically, there were exposure related increases in the incidences and severities of hematopoietic cell proliferation in the spleen at exposure concentrations of 5000 ppm or greater in males and females. Liver Kupffer cell erythrophagocytosis and hemosiderin pigmentation, as well as renal tubule hemosiderin pigmentation, occurred in exposed mice. Other lesions observed included hyperplasia of the transitional epithelial lining of the urinary bladder in male and female mice exposed to 2500 ppm or greater. There was degeneration of the nasal olfactory epithelium of the dorsal meatus in male mice exposed to 5000 ppm and greater and in female mice exposed 2500 ppm or greater.

Histopathological findings: neoplastic:

no effects observed

Details on results:

A NOAEL for the most sensitive parameter (hyperplasia of the urinary bladder transitional epithelium) for male mice was 625 ppm.

Dose descriptor:

NOAEL

Remarks:

bladder toxicity

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: hyperplasia of the urinary bladder transitional epithelium

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

bladder

Treatment related:

yes

Conclusions:

Based on results of this 13-week oral toxicity study of methyl ethyl ketoxime (MEKO) in mice, the major target organ was the spleen. Spleen weights were generally significantly greater than those of the controls in the male and female mice exposed to 10000 ppm. Microscopically, there were exposure related increases in the incidences and severities of hematopoietic cell proliferation in the spleen at exposure concentrations of 5000 ppm or greater in males and females. Methemoglobinemia and a responsive Heinz body anaemia were induced. Liver Kupffer cell erythrophagocytosis and hemosiderin pigmentation, as well as renal tubule hemosiderin pigmentation, occurred in exposed mice. Other lesions observed included hyperplasia of the transitional epithelial lining of the urinary bladder in male and female mice exposed to 2500 ppm or greater. There was degeneration of the nasal olfactory epithelium of the dorsal meatus in male mice exposed to 5000 ppm and greater and in female mice exposed 2500 ppm or greater. A NOAEL for the most sensitive parameter (hyperplasia of the urinary bladder transitional epithelium) for male mice was 625 ppm.

Reference 4

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13-weeks

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Results of this study are scientifically acceptable, the testing procedures and resulting data are sufficient and well documented. Although the study was not conducted according to GLPs, it was performed by a credible laboratory with an adequate study design. .

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

no

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, Wilmington, MA
- Weight at study initiation: Males 183-266 g; Females 150-191 g
- Fasting period before study: no
- Housing: Individually in elevated wire mesh cages
- Diet (e.g. ad libitum): Purina Laboratory Chow, ad libitum
- Water (e.g. ad libitum):ad libitum

Route of administration:

oral: gavage

Vehicle:

other: 0.5% carboxymethylcelluose

Details on oral exposure:

Solutions of MEKO were prepared in 0.5% carboxymethylcellulose so that the prescribed daily dose was administered by gavage with 2.0 mL of test solution/kg bw. The daily dose (2.0 mL/kg) was administered in two equally divided doses (.1.0 mL/kg) and was adjusted according to weekly individual body weights.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

5 days/week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

All animals were observed daily for mortality, morbidity, and signs of toxic and pharmacologic effects.

The body weight, food consumption, and clinical observations of each rat were recorded weekly throughout the study.

Hematology parameters (hematocrit, hemoglobin level, erythrocyte count, and total and differential leukocyte counts) were determined on 5 rats/sex/group at weeks 4, 8 and 13.

Blood chemistry parameters on 5 rats/sex/group were determined at weeks 4, 8 and 13. The following parameters were determined at each interval: fasting glucose, blood urea nitrogen, total protein, total bilirubin, serum glutamic-pyruvic treansaminase, alkaline phosphatase, and serum electrophoresis. The following parameters were also determined at week 13: serum glutamic-oxaloacetic transaminase, serum albumin, serum sodium, serum potassium, serum chloride, serum calcium, and carbon dioxide. Erythrocyte and plasma cholinesterase levels were determined at weeks 4, 8 and 13 on an additional set of 5 rats/sex/group.

Urine analyses (specific gravity, pH, ketones, total protein, bilirubin and microscopic examination of the sediment) were performed on pooled samples from 5 rats/sex/group at weeks 4, 8 and 13.

Sacrifice and pathology:

Necropsies were performed on all animals that died during the course of the study and on all rats sacrificed at termination, week 13. Prior to sacrifice, all rats were fasted for 24 hours and the terminal body weights were recorded. The following organs of each rat were weighed and the organ/body weight ratios determined: liver, spleen, heart, kidneys and testes with epididymides (prior to fixation) and thyroid and adrenals (after fixation).

All preserved tissues from the control and high-dose groups at week 13 were stained and examined microscopically. In addition, sections of the liver, spleen and kidneys were examined microscopically from all the animals of the low- and mid-dose groups.

Additional histopathology was conducted on the testes of all male animals in the study (refer to 1988 reference listed above, addendum to report). Sections of testes with epididymides were prepared from formalin-preserved testes for all low- and mid-dose animals and slides were prepared from existing blocks from all control and high-dose animals. Each section was examined microscopically.

Liver sections from this study were reexamined in order to determine if MEKO caused similar lesions indicative of hepatotoxicity which were observed for the high dose male rats in a subchronic 90-day oral toxicity study with acetone oxime (refer to 1990 reference listed above).

Statistics:

Statistical analyses of food consumption data, clinical laboratory data (hematology and blood chemistry), terminal body weights, organ weights, and organ/body weight ratios were performed by Bartlett's test of homogeneity of variances and the one-way classification analysis of variances. When differences were noted in the analysis of variances, Scheffe's method for judging all contrasts and Student's t-test were employed.

Statistical analysis of the mean body weight data at weeks 0, 4, 8 and 13 was performed using the F-test (analysis of variance) and the Student's t-test. When variances differed significantly, Student's t-test was approximately modified (t') and Cochran's approximation was utilized.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Effects on blood parameters indicative of hemolytic anemia and compensatory hematopoiesis, as well as extramedullary hemosiderosis in the spleen were observed. A NOAEL was not established. In the high dose group (225 mg/kg bw/day) the degree of hemolytic anemia is relatively serious. Although not all the hematological parameters are statistically significant at 25 mg/kg/day, there is still a clear trend observable at this dose. Therefore, the NOAEL is below 25 mg/kg bw/day. Extrapolation on basis of dose-response indicates a NOEL at about 10 mg/kg bw/day. Based on interpolation between the 25 and 75 mg/kg bw/day (low dose and mid-dose) it seems that all features of hemolytic anemia with compensatory hematopoiesis (including hematological parameters and pigment accumulation) are clearly present at 50 mg/kg bw/day.

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Dose related effects on organ weights of spleen and liver were seen.

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Results of the microscopic evaluation of sections in testes with epididymides (refer to addendum to report, 1988) revealed a reduction in spermatogenic activity in testes from 2 rats each in the control and low-dose groups, 3 rats in the mid-dose group and 5 rats in the high-dose group. The reduction in spermatogenic activity was generally more severe in the high-dose rats. Sections of testes/epididymides from the remaining 20 high-dose rats revealed normal spermatogenic activity and were comparable in appearance to those from rats of the control and lower-dose groups.

Results of the microscopic reexamination of liver sections found a dose-related accumulation of pigment and increased incidence of extramedullary hematopoiesis in the liver of male and female rats dosed with 25, 75 and 225 mg MEKO/kg/day as compared to control rats of both sexes. A no observed effect level was not present since minimal degrees of pigment accumulation and/or hematopoiesis was present in the liver of both sexes of rats at the low dose. Hepatocellular changes indicative of direct hepatic toxicity similar to those induced by acetone oxime exposure were not observed in either sex.

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

LOAEL

Remarks:

blood effects

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on haemolytic anaemia and compensatory haematopoiesis.

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

haematopoietic

Organ:

blood

Treatment related:

yes

Dose response relationship:

yes

Conclusions:

Oral administration of methyl ethyl ketoxime (MEKO) by gavage for 13-weeks at dose levels of 0, 25, 75 or 225 mg/kg bw/day caused dose-related effects on organ weights of the spleen and liver. Effects on blood parameters indicative of hemolytic anemia and compensatory hematopoiesis, as well as extramedullary hemosiderosis in the spleen were observed at 25 mg MEKO/kg bw/day or greater. Based on these results, the NOAEL was <25 mg/kg bw/day and the LOAEL was 25 mg/kg bw/day. A reduction in spermatogenic activity in a few male rats, observed after addtional histopathologic examination of the testes, was not considered related to MEKO treatment, but rather incidental to treatment. Histopathologic reexamination of liver sections from this study did not find hepatocellular changes indicative of direct hepatic toxicity similar to those induced by acetone oxime.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

25 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 1-2) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VII, 8.6, of Regulation (EC) No 1907/2006.

System:

haematopoietic

Organ:

blood

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

54 mg/m³

Study duration:

chronic

Experimental exposure time per week (hours/week):

30

Species:

rat

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VII, 8.6, of Regulation (EC) No 1907/2006.

System:

hepatobiliary

Organ:

liver

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

This study was designed to further investigate the effect of MEKO on the olfactory epithelium and establish a NOAEC, determine if reversible with recovery phase. The mouse was chosen since it appears to be more sensitive to this effect than the rat. Liver peroxisome proliferation and glutathione content were also evaluated.

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Char1es River Laboratories, Portage, Michigan
- Age at study initiation: 7 weeks
- Weight at study initiation: 27.6 (25.0-30.0) g
- Fasting period before study: no
- Housing: Animals were doubly housed in suspended, stainless steel, wire mesh cages during the first week of the acclimation period and individually housed during the remainder of the acclimation period and all other non-exposure periods. During exposures, animals were individually housed in
wire mesh, stainless steel cages.
within a 6000 l i t e r glass and
stainless steel exposure chamber
- Diet (e.g. ad libitum): Certified mash 1aboratory diet (Purina Mills Rodent Diet #5002). Fresh food presented as required.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 (average)
- Humidity (%): 46 (average)
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

Chamber environmental conditions averaged 21°C and 49% relative humidity. Mass and particle size distribution measurements during chamber trials indicated that there was no measurable amount of MEKO likely to be present as an aerosol.

Pre-Study Tria1s : Trial s were performed to evaluate the optimal set o f equipment and operating conditions to generate a stable atmosphere at the targeted exposure levels.

Chamber Operation : The exposure chambers were operated dynamically under slight positive pressure. The chamber's airflow rate, total flow rate, time for air change and 99% equilibrium time (T99) for each group were determined. The chamber size and airflow rate was considered adequate to maintain the animal loading factor below 5% and oxygen above 19%. The chamber was exhausted through a system consisting of a coarse filter , a
HEPA filter and into an incinerator. Recordings o f chamber temperature, relative humidi ty, airflow rate and static pressure were made every ha1 f-hour during exposure. The volatilization chamber consisted of two concentric glass tubes sealed at the ends. The outer tube had ports for the delivery of MEKO and nitrogen and the exhaust of the vapor-laden atmospheres. The inner glass tube was equipped with a glass coil spiraling down around the outside of the tube and an internal heating element. MEKO was pumped onto the top of the glass coil, where it flowed down and around the heted tube. Heated nitrogen was introduced a the bottom of the volatilization unit to flow upward, counter to the flow of MEKO, to maximize volatilization. The vapors were generated under nitrogen to get the vapor-laden atmospheres near or below the lower-explosion-limit prior to mixing with air. The output of the volatilization chamber was connected to the entry turret of the exposure chamber, where it mixed with air prior to entrance into the animal compartment of the chamber. The temperature of the volatilization unit was measured using an alarmed monitoring system.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Determination of the MEKO exptsure level s were made using a MIRAN Ambient Air analyzer equipped with a strip chart recorder. The test atmospheres were drawn at one hour intervals through the MIRAN and measurements were recorded at least hourly during the exposures from the normal sampling portal and once during exposures from the distribution sampling portal. The exposure levels were determined by comparison of the measured absorbance to a calibrated response curve constructed using the same instrument settings.

Particle size distribution measurements were performed once during each trial for the chambers and room ai r using a TSI Aerodynamic Particle Sizer equipped with a diluter. The samples were drawn for 20 seconds at a rate of 5 liters per minute. The mass median aerodynamic diameter, geometric standard deviation and percent of particles 11.0, 54.0 and 510 microns were calculated. A computer was used to program the system to the appropriate settings prior to sampling. The particle size distribution s were calculated by the computer and printed out following an analysis of an assumed lognormal distribution .

Duration of treatment / exposure:

Main study: 1, 2, 4 or 13 weeks.
Satellite groups: additional recovery period of 4 or 13 weeks

Frequency of treatment:

6 h/day, 5 days/week

Dose / conc.:

3 ppm (analytical)

Remarks:

± 0.1 ppm (10.8 mg/m³)

Dose / conc.:

10 ppm (analytical)

Remarks:

± 0.3 ppm (36 mg/m³)

Dose / conc.:

30 ppm (analytical)

Remarks:

± 1.0 ppm (108 mg/m³)

Dose / conc.:

100 ppm (analytical)

Remarks:

± 2.0 ppm (360 mg/m³)

No. of animals per sex per dose:

Main study: 10 males/dose/interval (exposures for 1, 2, 4 or 13 weeks)
Satellite groups: 5 males/dose/interval (satellite animals were removed after 1, 2, 4 or 13 weeks and allowed to recover for 4 or 13 weeks)

Control animals:

yes

Details on study design:

- Dose selection rationale: Exposure levels were selected by the Sponsor on the basis of available toxicity data.
- Rationale for selecting satellite groups: In a previous inhalation oncogenicity study of MEKO in mice and rats (in which exposures to MEKO were at concentrations of 15, 75 and 374 ppm for 6 hours/day, 5 days/week for 2 years), MEKO was found to be a liver oncogen in male mice at 374 ppm and male rats at 75 ppm. In addition, olfactory degeneration was seen in both rats and mice in all MEKO exposure groups. For this reason, the current study was designed to investigate the time course of development and reversibility of the degeneration seen i n the olfactory region of the nasal turbinates. Only male mice were used in this study because there was no gender difference in the response and the mice seemed. to be more sensitive than the rats to this effect.
- The 3 and 10 ppm MEKO exposures were chosen because 15 ppm was not a no observed effect level in the previous chronic study. The higher exposure levels of 30 and 100 ppm, along with the 1-,2-, 4- or 13 week exposure durations and the 4 and 13 week recovery after each exposure duration were selected in order to evaluate the time course for development of the olfactory epithelial lesion and its reversibility.

Observations and examinations performed and frequency:

Mortality and Gross Signs of Toxicologic or Pharmacologic Effects: Twice daily.

For Abnormal Signs: Daily. Animals were observed as a group once during each exposure.

Detailed Physical Examinations: Pretest and weekly thereafter.

Body Weight: Twice pretest, weekly during study period and prior to scheduled sacrifice.

Sacrifice and pathology:

Post Mortem Examination: Microscopic examination was performed on the nasoturbinates of all animals. Degenerative changes in each individual section examined were scored for severity for a relative comparison among the exposure groups. Severity scores were based on the subjective assessments of degree of degeneration (decrease in thickness) and the approximate percentage of the olfactory epithelium affected. The severity scores defining the approximate extent of tissue involvement were: Minimal, up to 2%; Slight, from 2 to 10%; Moderate, from 10 to 30%; Moderately Severe, from 30 to 70%; and Severe, over 70%.

Generalized maps were prepared to show the distribution of morphological changes in the olfactory epithelium. A comprehensive set of diagrams suitable fo recording the location and distribution of tissue responses for each transverse sectional level of the nose was used for the mapping procedure.

Peroxisome Proliferation: For the animals sacrificed after 13 weeks of exposure, potential peroxisome proliferation in the liver was evaluated using electron microscopy o f selected liver samples and by examining the effect of MEKO on mouse hepatic peroxisomal fatty P-oxidation.

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):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At the end of the 1, 2, 4 and 13 week exposure periods, degeneraton of the olfactory epithelium lining the dorsal meatus was seen in the anerior region of the nasal cavity. In a few instances, the olfactory epithelium covering the tips of the nasoturbinal scrolls projecting into the dorsal region of the nasal cavity was also degenerated. Large areas of olfactory epithelium lying laterally and posteriorly were unaffected. In general, approximately 10% or less of the total olfactory tissue was affected. In several instances, the degenerated olfactory epithelium was reepithelialized by squamous/squamoid and /or respiratory types of epithelium. Degeneration, which was dose-related in incidence and severity, was seen in mice exposed to 30 and 100 ppm after 1 week of exposure and in several mice exposed to 10 ppm after 13 weeks of exposure. The incidence and severity of the degeneration present after 1 week of exposure did not increase with the longer exposures. The olfactory degeneration was reversible. Recovery was complete within 4 weeks following exposures at 10 ppm and nearly complete withn 13 weeks after exposures at 30 and 100 ppm.

Histopathological findings: neoplastic:

no effects observed

Details on results:

The 3 ppm concentration was considered the NOAEC.

Dose descriptor:

NOAEC

Effect level:

10.8 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: olfactory epithelium degeneration in the nasal cavity

Critical effects observed:

yes

Lowest effective dose / conc.:

36 mg/m³ air (analytical)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Conclusions:

Under the conditions of this study, inhalation exposure to MEKO at concentrations of 10, 30 or 100 ppm for 6 hours/day, 5 day/week for 13 weeks produced minimal to moderately severe olfactory epithelium degeneration in male CD-1 mice. The incidence and severity of the degeneration was concentration dependent and not progressive over time with continued exposure. The lesions were localized to the olfactory epithelium lining the dorsal meatus in the anterior portion of the nasal cavity. Large areas of olfactory epithelium laterally and posteriorly appeared unaffected. No effects to olfactory epiithelium were found at 30 ppm MEKO in > 50% of the animals, indicating that only the most sensitive animals responded at this relatively high level of exposure. The degeneration of the olfactory epithelium following exposure to MEKO is not widespread but limited primarily to cells in the dorsal anterior portion of the nasal cavity. This represents approximately 10% of the olfactory epithelium lining the nasal cavity. The effect was reversible with cessation of exposure with complete recovery observed within 4 weeks at 10 ppm and nearly complete recovery after 13 weeks at the higher concentrations. The low incidence of lesions that occurred at 10 ppm only after 13 weeks of exposure suggests that this exposure concentration is near the threshold for the olfactory effects of MEKO. The NOAEC was considered to be 3 ppm.
Under the exposure conditions of this study, MEKO did not induce hepatic peroxisome proliferation nor did it cause any ultrastructural changes in liver cells after 13 weeks of exposure at concentrations up to 100 ppm. However, significant increases in levels of hepatic non-protein sulphydryl groups (primarily reduced glutathione) were measured following MEKO exposures at 30 and 100 ppm.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

10.8 mg/m³

Study duration:

subchronic

Species:

mouse

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 1) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VII, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral route of exposure

Gavage and drinking water studies with durations of 4 and 13 weeks have been conducted with rats and one 13-week drinking water study has been conducted in mice. A common effect observed in these studies is haematological effects such as anaemia. Degeneration of the nasal epithelium was seen in both drinking water studies.

 

In one 13-week gavage study with rats (Allied Chemical Corporation, 1977), 0, 25, 75 and 225 mg/kg bw/day the test substance in 0.5% carboxymethylcelluose were submitted to groups of 10 male and 10 female rats 5 days/week for 13 weeks. Dose-related effects on organ weights of spleen and liver were seen. Effects on blood parameters indicative of haemolytic anaemia and compensatory haematopoiesis, as well as extramedullary haemosiderosis in the spleen were observed.  In the high dose group (225 mg/kg bw/day) the degree of haemolytic anaemia was relatively serious. Although not all parameters were statistically significant at 25 mg/kg bw/day, there was a clear trend observable at this dose. A NOAEL was not established. A LOAEL of 25 mg/kg bw/day (lowest dose tested) was established, based on haemolytic anaemia.

 

A 13-week drinking water study, performed according to the protocol similar to EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents) Guideline was conducted with rats and mice (NTP, 1999a). In the rat study, animals received 0, 312, 625, 1250, 2500 and 5000 ppm the test substance (corresponding to actual ingested doses of 0, 25, 50, 100, 175 and 280 mg/kg bw/day (males) and 0, 30, 65, 120, 215 and 335 mg/kg bw/day (females)). The NOAEL was reported to be 312 ppm (corresponding to actual ingested doses of 25 and 30 mg/kg bw/day in males and females, respectively). The LOAEL of 625 ppm (50 and 65 mg/kg bw/day in male and female rats, respectively) was based on erythrotoxicity. The incidence and severity of haematopoietic cell proliferation in the spleen showed dose-related increases at = 50 mg/kg bw/day in males and = 65 mg/kg bw/day in females. Haematopoietic proliferation in the bone marrow was also significantly increased at these doses. Liver Kupffer cell erythrophagocytosis, haemosiderin pigmentation, and renal tubule haemosiderin pigmentation were also observed. Haematology indicated methaemoglobinemia and a responsive Heinz body anaemia. Final mean body weights and body weight gains were smaller at doses of = 215 mg/kg bw/day in the females when compared with the controls. In males, mean body weight gains were lower at doses of = 100 mg/kg bw/day. Liver and spleen weights were significantly greater than those of the controls in males at = 100 mg/kg bw/day and in females at = 120 mg/kg bw/day. Kidney weights were significantly increased in males at 280 mg/kg bw/day and in females at = 120 mg/kg bw/day.Degeneration of the nasal epithelium was observed at 2500 ppm (215 and 275 mg/kg bw/day) and higher.

 

In the 13-week study with mice (NTP, 1999a), animals received 0, 625, 1250, 2500, 5000 and 10000 ppm (corresponding to 0, 110, 200, 515, 755 and 1330 mg/kg bw/day (males) and 0, 145, 340, 630, 1010 and 3170 mg/kg bw/day (females)) the test substance in drinking water for 13 weeks. Mean body weights and body weight gains were lower at the highest dose than those of the controls. Also, spleen weights were generally significantly greater than those of the controls in both sexes at the highest dose. Microscopically, exposure-related increases in the incidence and severity of haematopoietic cell proliferation in the spleen were seen at 755 mg/kg bw/day in males and 1010 mg/kg bw/day in females or greater. Liver Kupffer cell erythrophagocytosis, haemosiderin pigmentation, and renal tubule haemosiderin pigmentation occurred in exposed mice. Other lesions included hyperplasia of the transitional epithelial lining of the urinary bladder in male and female mice at 515 mg/kg bw/day in males and 630 mg/kg bw/day in females or greater. The nasal olfactory epithelium of the dorsal meatus was degenerated in male mice exposed to 755 mg/kg bw/day and in female mice exposed to 630 mg/kg bw/day and above. Haematology indicated a methaemoglobinemia and a responsive Heinz body anaemia. A NOAEL for the most sensitive parameter (hyperplasia of the urinary bladder transitional epithelium) for male mice was 110 mg/kg bw/day.

In addition, to study the sub-chronic neurotoxicity of the test substance in rats, rats were dosed 5 days/week by gavage for 13 weeks with 40, 125, or 400 mg/kg bw/day (Schulze et al., 1993a). Neurobehavioral assessments, clinical observations, periodic body weights, and food consumption, selected organ weights, gross pathological, and neuropathological examinations were utilized to detect treatment- related effects. To check for neurotoxicity the standard "Functional Observational Battery", motor-activity data and neurohistopathology were performed. Acrylamide was used as a positive control.

At dose levels of 40 and 125 mg/kg bw/day, the test substance 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 haematology parameters. Transient neurobehavioral changes (cage removal, handling, posture, gait, arousal, salivation, approach response, rearing responses, and aerial righting) occurred following the test substance doses of 400 mg/kg bw/day, were noted immediately after dosing, and had resolved by the next day prior to administration of the next dose. 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.

 

In a 4-week gavage study (Ministry of Health & Welfare (MHW), Japan, 1996), groups of 7 male and female Fischer rats received 0, 4, 20 and 100 mg/kg bw/day the test substance in olive oil daily for 28 days. A post-exposure follow up was conducted on seven animals per sex of the control and seven high dose animals per sex that were untreated for an additional 14 days. Absolute and relative spleen weights were increased in females administered 20 mg/kg bw/day and in males given 100 mg/kg bw/day and above. Relative lung weights were increased in high dose males, and relative heart weights were increased in high dose females. Autopsy revealed hypertrophy of the spleen in mid and high dose males and in high dose females. Congestion, increase in extramedullary haematopoiesis, and an increase in haemosiderin granules were observed in the spleens of mid and high dose animals of both sexes. Kupffer cell hypertrophy with phagocytosed haemosiderin granules in the liver was observed in high dose males and in the mid and high dose females. Extramedullary haematopoiesis in the liver and deposition of lipofuscin-like substance in the renal tubular epithelium were observed in high dose animals of both sexes. The haematological examination revealed an increase in reticulocyte ratio in males and females administered 20 mg/kg bw/day and above and an additional increase in the platelet count in females at the same dose level and above.  Decreases were seen in the red blood cell count, haematocrit value and haemoglobin concentration in males given 100 mg/kg bw/day and in females given 20 mg/kg bw/day. Mean corpuscular volume, mean corpuscular haemoglobin and white blood cell counts were increased in both sexes at the highest dose. Other blood chemistry showed an increase in the potassium levels at the highest dose in both sexes. Also, in males at the highest dose, increases in the albumin/globulin ratio and albumin fraction, a decrease in the A1-globulin fraction, and increases in total bilirubin and total cholesterol were observed. Most of the observed changes disappeared or were decreased at the end of the recovery period. However, based on several effects seen at 20 mg/kg bw/day and higher, the NOAEL for males and females was considered to be 4 mg/kg bw/day. This study indicates that the blood system is a target of butanone oxime. However, little information was given regarding incidence or severity of these findings, and most of the finding were reversible.

 

In another 4-week gavage study (Pharmaco-LSR, 1995a), Fischer 344 male rats (15 in each group) were dosed with 0, 250, and 500 mg/kg bw/day the test substance in water. The study was designed to assess potential peroxisome proliferation, changes in hepatic glutathione levels, and serum testosterone levels after administration of the test substance. Control animals (15/group) received distilled water (negative control), 0.5% methylcellulose (vehicle control) or clofibric acid, 250 mg/kg bw/day (positive control), at the same dose volume as administered to the treated animals.

Under the conditions of this study, the test substance did not produce any significant hepatic peroxisome proliferation in the rat, as indicated by a lack of effect on palmitoyl-CoA oxidation and electron microscopy. The potential responsiveness of the animals used in this study was confirmed by the marked induction of palmitoyl-CoA in the rats treated with clofibric acid. No effects on testosterone levels were found. Although peroxisome proliferation did not occur, significant increases in hepatic glutathione (primarily reduced glutathione) were observed in rats given 250 and 500 mg/kg bw/day for 14 days. This lack of an effect after 7 days of dosing correlated with the hepatocellular hypertrophy which was seen microscopically after 14 and 28 but not 7 days of treatment.

 

 

Inhalation route of exposure

 

In a 13-week inhalation study in CD-1 mice (Pharmaco LSR, 1995b), groups of 10 male mice were exposed to 0, 3, 10, 30 and 100 ppm (0, 10.8, 36, 108 and 360 mg/m³) for 1, 2, 4 and 13 weeks. Satellite animals (5/dose/interval) were removed after 1, 2, 4 or 13 weeks and allowed to recover for 4 or 13 weeks. A NOAEC of 3 ppm (10.8 mg/m³) was established based on olfactory effects in a small area of tissue in the nose at doses of 10 ppm (36 mg/m³) and above. These effects were minimal at lower doses and moderately severe at higher doses. After cessation of exposure, the effects showed reversal to varying degrees, depending on dose. (It should be noted that the nasal morphology and respiratory physiology of the rodent is different from humans such that higher local exposure of the olfactory epithelium is expected to occur in rodents as compared to humans at equivalent airborne concentrations of the test substance.)

 

A 4-week study in rats administered the test substance vapor at 25, 100 and 400 ppm (90, 360 and 1440 mg/m³) for 6 hr/day 5 days/week revealed blood system effects indicative of haemolytic anaemia and compensatory haematopoiesis at 1440 mg/m³. A NOAEC of 90 mg/m³ was identified based on increased methaemoglobin in female rats and increased absolute or relative organ weights (liver and spleen) of male and female rats (Bio/Dynamics Inc., 1990). In mice exposed in the same regimen, a NOAEC of 360 mg/m³ was determined based on slight increases in methaemoglobin and increased spleen and adrenal weights (Bio/Dynamics Inc., 1990).

 

In another 4-week inhalation study in rats, conducted by Dow Corning Corporation (1983), groups of 20 rats were exposed to vaporised concentrations of 0, 60, 283, 533, or 714 ppm (210, 1020, 1920 or 2570 mg/m³) the test substance 6 hours/day, 5 days/week for 4 weeks. Examination of abnormal behaviour and gross external lesions, body weights, haematology, blood chemistry, urinalysis, and gross and histopathology were conducted. There were significant decreases in serum glucose levels and red blood cells at the two highest exposure concentrations and decreases in blood creatinine, SAP, Na, and K, at the highest concentration. Statistically-significant increases in blood mean corpuscular volume, mean corpuscular haemoglobin, and reticulocyte, were observed at the two highest doses. Also, haemosiderosis in the spleen (possibly due to increased breakdown of red blood cells) was seen in the high dose group. In addition, changes in brain and spleen weights were observed at the top two doses. A NOAEC of 1020 mg/m³ was established. This study however may have been compromised by a viral lung infection that occurred in all groups, including the controls, and was therefore disregarded. 

 

In addition, a carcinogenicity inhalation study with rats and mice is available, which is summarised in the section on carcinogenicity (Newton et al., 2001). CD-1 mice (50/sex/group) and F-344 rats (50/sex/group) were exposed 6 h/day, 5 days/week for 18 (mice) or 26 months (rats) via whole-body inhalation exposures to target the test substance vapor concentrations of 0, 15, 75, and 375 ppm (corresponding to 0, 54, 270 and 1350 mg/m³). Satellite groups of rats and mice (10/sex/group/interval) were exposed for 12 months (mice) and 3, 12, or 18 months (rats) to evaluate chronic toxicity. Methyl ethyl ketone (MEK), a possible hydrolysis product of the test substance, was present at less than 1%.

In rats, effects on blood parameters indicative of haemolytic anaemia and compensatory haematopoieseis were seen at 1350 mg/m³ at 3 months and 12 months but not at 18 months in males and not at 26 months in either sex. Effects to the spleen (increased organ weight, extramedullary haematopoiesis and haemosiderosis) were seen at the same dose level. Spleen weights were increased in both sexes at 3 and 12 months, at 18 months in females only, but were not increased in either sex at 26 months. Histopathological effects persisted at the same time points as the spleen weights, but could not be accurately determined at termination due to leukaemia. Liver weights were also increased at the highest dose. At 18 and 24 months as well as for all unscheduled deaths, an increased incidence of red/tan discoloration of the liver was observed. Nodules and masses could also be seen macroscopically in the liver at 24 months. Effects observed in the liver of the rats included decreases in the incidence of both peribiliary fibrosis and hyperplasia/proliferation of the biliary duct, an increase of spongiosis hepatitis in males, and an increase in the incidence of intracytoplasmic vacuoles and hepatocellular basophilic foci. The effects on the liver were generally most profound in the high-exposure groups and, with the exception of the spongiosis hepatitis, occurred in both sexes. An increase in hepatocellular adenomas occurred in the male rats at 270 and 1350 mg/m³, and hepatocellular carcinomas in the male rats at 1350 mg/m³.

A high incidence of cataracts and corneal dystrophy occurred in both control and exposed rats, with an earlier appearance and slightly higher incidence for these ocular lesions in exposed animals compared to controls. Degenerative and reparative changes of the olfactory epithelium in the nasal turbinates, primarily limited to the dorsal meatus, occurred in rats at 270 and 1350 mg/m³).

The effects on the blood were not as clear for mice as for rats. There was an increase in methaemoglobin at 12 months only in males. Other erythrocyte parameters were generally unaffected in both sexes and spleen weights and spleen pathology were not affected (there was no 3 month sacrifice and the study was terminated at 18 months). In addition, in the mice, liver changes included increased incidences of pigment in reticuloendothelial cells, centrilobular hypertrophy, granulomatous inflammation, and a slightly increased incidence of necrosis (270 and 1350 mg/m³). An increase in hepatocellular carcinomas occurred in male mice at 1350 mg/m³. Degenerative and reparative changes of the olfactory epithelium in the nasal turbinates, primarily limited to the dorsal meatus, occurred in mice at 54, 270 and 1350 mg/m³.

Based on the results of the study, a NOAEC for carcinogenicity was set to 270 mg/m³ for male rats and mice and 1350 mg/m³ for female rats and mice. A NOAEC for toxicity was set at 54 mg/m³ for rats, based on increased incidence of hepatocellular adenomas (males only) and degenerative and reparative changes of the olfactory epithelium in nasal turbinates, while for mice the same concentration level was a LOAEC based on the effects on the olfactory epithelium. In conclusion, the study chosen as key study is the carcinogenicity study, which has the longest duration (26 months) and the lowest NOAEC.

 

Taken together, there are several oral and inhalation studies reporting effects indicative of anaemia at doses sufficiently low to justify classification. These effects have been seen in rats and rabbits. Although full descriptions of the severity of the effects seen on the blood system are not available, the consistent nature of the findings reported is a concern. The CLP guidance provides examples of effects fulfilling classification criteria for substances inducing anaemia. For the test substance, there are several aspects of its toxicity to the blood system that can be related to these criteria, supporting classification for STOT RE:
-Premature deaths in anaemic animals that are not limited to the first three days of treatment in the repeated dose study. (Mortality during days 0-3 may be relevant for acute toxicity.)
-Clinical signs of hypoxia, e.g. cyanosis, dyspnoea, pallor in anaemic animals that are not limited to the first three days of treatment in the repeated dose study.
-Reduction in functional Hb at ≥ 20 % due to a combination of Hb reduction and MetHb increase.
-Marked increase of haemosiderosis in the spleen, liver or kidney in combination with other changes indicating significant haemolytic anaemia (e.g. a reduction in Hb at ≥ 10 %) in a 28-day study.
-Significant increase in haemosiderosis in the spleen, liver or kidney in combination with microscopic effects like necrosis, fibrosis or cirrhosis.

The doses at which these effects were observed were sufficiently low to match the criteria for a category 2 classification.

Justification for classification or non-classification

The available data on repeated dose toxicity of the test substance meet the criteria for classification as STOT RE 2 (H373) according to Regulation (EC) 1272/2008.