Acetone oxime

Toxicity to reproduction

Administrative data

Endpoint:

toxicity to reproduction

Remarks:

other: subchronic

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

September 20, 2004 - June 1, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test method according to OECD 408. GLP study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-methylpentan-2-one oxime

EC Number:

203-298-2

EC Name:

4-methylpentan-2-one oxime

Cas Number:

105-44-2

IUPAC Name:

4-methylpentan-2-one oxime

Details on test material:

- Name of test material (as cited in study report): MIBKO, methyl isobutyl ketoxime
- Physical state: liquid
- Analytical purity: 99.9%
- Lot/batch No.: 03120011A
- Expiration date of the lot/batch: 1 July 2005
- Storage condition of test material: At 4°C and in dry conditions

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent, England
- Age at study initiation: 44 to 48 days
- Weight at study initiation: 196 to 275 g for males and 150 to 216 g for females
- Fasting period before study:
- Housing: Five of one sex per cage, stainless steel body cages with a stainless steel mesh lid and floor.
- Diet (e.g. ad libitum): Ad libitum (except when urine was being collected and overnight before routine blood sampling).
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23 ºC
- Humidity (%): 40-70 %
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hrs dark/12 hrs light).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Starting with the lowest concentrated formulation, the required volume of test substance was measured into a mixing jar to which was measured the required volume of vehicle. The formulation was then mixed using a spatula to remove any test substance adhering to the jar, then mixed with a whirlimixer for approximately one minute. Each subsequent dose concentration was formulated similarly, in ascending order. Formulation was
stirred continuously using a magnetic stirrer. Doses were prepared weekly and split into daily aliquots.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0, 2.5, 7.5, 25 mg/mL
- Amount of vehicle (if gavage): 2 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each formulation prepared for administration in Weeks 1 and 12 of treatment were analysed for achieved concentration of the test substance. The analytical procedure, gas liquid chromatographyc analysis with flame ionisation detection (FID), was validated for MIBKO with respect to linearity of detectorresponse, precision of injection, specificity of chromatographic analysis, limit of detection, accuracy and precision. The homogeneity and stability of MIBKO in dried corn oil formulations were confirmed at nominal concentrations of 2.5 mg/ml and 25 mg/ml during storage at ambient temperature for 2 days and refrigeration for up to 15 days. The mean concentrations of MIBKO in the test formulations analysed during the study were within 10% of nominal concentrations, confirming the accuracy of formulation.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
5, 15, 50 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

Mainstudy (13 weeks): 10 animals per sex and per dose.
Recovery phase (13 weeks + 4 weeks recovery): 5 animals per sex and per dose.
Total of 120 animals.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dosages were selected based on previous work with the laboratory. Results from these studies indicated that a level of
150 mg/kg/day or above would not be tolerated over 13-weeks of treatment and it was predicted that treatment at 50 mg/kg/day for 13 weeks would result in tolerable toxicity for each test substance.
- Post-exposure recovery period in satellite groups: 4 weeks

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Timie schedule: at least twice daily
- Observations: evidence of ill-health or reaction to treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and recovery
- Observations: detailed physical examination and arena observations were performed on each animal.

BODY WEIGHT: Yes
- Time schedule for examinations: one week before treatment commenced (Week -1), on the day that treatment commenced (Week 0), weekly throughout the treatment and recovery periods and before necropsy.

FOOD CONSUMPTION:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started (Week -1), and each week throughout the treatment and recovery periods. From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Fluid intake was assessed by daily visual observation during the study. As there was no observed effect on water intake no precise measurements of fluid intake were carried out.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment commenced and during week 12. No examination needed at the end of the recovery because no treatment related changes observed at Week 12.
- Dose groups that were examined: All animals of 0 (control) and 50 mg/kg bw/day doses.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Weeks 5 and 13 of treatment (all parameters), Week 4 of recovery (red and white blood cell parameters).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight starvation.
- How many animals: All animals
- Parameters checked: Haematocrit (Hct), Haemoglobin (Hb), Erythrocyte count (RBC), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Total white cell count (WBC), Differential WBC count, Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (PLT), Abnormal morphologies, Prothrombin time (PT), Activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Weeks 5 and 13 of treatment (all parameters), Week 4 of recovery (few parameters based on previous results)
- Animals fasted: Yes, overnight starvation.
- How many animals: All animals
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total bilirubin (Bili), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Inorganic phosphorus (Phos), Total protein (Total Prot), Albumin (Alb), Albumin/globulin ratio (A/G Ratio). 4 week recovery checked parameters: phosphorus for both sexes, bilirubin for males, cholesterol, total protein, albumin and A/G ratio for females.

URINALYSIS: Yes
- Time schedule for collection of urine: Week 13 of treatment and Week 4 of recovery.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes.
- Parameters checked: Appearance, Volume (Vol), pH, Specific gravity (SG), Protein (Prot), Glucose (Gluc), Ketones (Keto), Bile pigments (Bili), Haem pigments (Blood), Microscopic examination of the urine sediment (Crystals (Cryst), Epithelial cells (Epi), Leucocytes (Leuc), Erythrocytes (RBC), Casts, Spermatozoa and precursors (Sperm), Other abnormal components (Abn)).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During weeks 5 and 12
- Dose groups that were examined: all recovery phase animals and the first five surviving main study animals from all groups.
- Battery of functions tested: sensory activity / grip strength / motor activity

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
All animals were subject to a detailed necropsy (at the end of treatment and recovery period).
Observations: All external features and orifices. The cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After
ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded.
The requisite organs were weighed and external and cut surfaces of the organs and tissues were examined as appropriate.
ORGAN WEIGHTS: Adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus with cervix (bilateral organs were weighed together).

HISTOPATHOLOGY: Yes
HISTOLOGY: For those animals specified in the Pathology section, the relevant tissues were subject to histological processing.
Those tissues subjected to histological processing included: Adrenals, Brain, Femur with joint, Heart, Kidneys, Liver, Lungs, Spinal cord, Sternum, Stomach, Thyroid, Uterus.
PATHOLOGY (microscopic examination): All tissues preserved for examination were examined for all animals of control and 50 mg/kg bw/day groups: adrenals, aorta-thoracis, brain, caecum, colon, duodenum, epydidymides, femurs with joint, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes, mammary area-caudal, oesophagus, ovaries, pancreas, pituitary, prostate, rectum, salivary glands, seminal vesicles, skin, spinal cord, spleen, sternum, stomach, testes, thymus, thyroid with parathyroids, trachea, urinary bladder, uterus und cervix. Tissues reported at macroscopic examination as being grossly abnormal were examined for all Main and Recovery animals. The microscopic examination was extended to the kidneys in males and spleen in both sexes for all animals of recovery phase.

Statistics:

For categorical data, the proportion of animals was analysed using Fisher’s Exact test (Fisher 1973). For grip strength and motor activity data, and for continuous data, Bartlett’s test was first applied to test the homogeneity of variance and treated groups were then compared with the control group, incorporating adjustment for multiple comparisons where necessary. The following sequence of statistical tests was used for grip strength and motor activity, bodyweight, food consumption, organ weight and clinical pathology data: If 75% of data were the same value, groups were compared using a Mantel test for a trend in proportions and also pairwise Fisher's Exact tests for each group against de control. If Bartlett's test for variance homogeneity was not significant at the 1% level, parametric analysis was applied. If the F1 test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 test was significant, Dunnett's test was performed instead. If Bartlett's test was significant at the 1% level, logarithmic and square-root transformations were tried. If Bartlett's test was still significant, non-parametric tests were applied. If the H1 test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 test was significant, Steel's test was performed instead. For organ weight data analysis of covariance was performed. If the within group relationship between organ weight and bodyweight was significant at the 10% level, then the treatment comparisons were made on adjusted group means. Any further pairwise comparison was performed using Student’s ‘t’ test. Significant differences between Control and treated groups were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Details on results (P0)

CLINICAL SIGNS AND MORTALITY:
There were no deaths during the treatment period.
There were no post dose signs observed during the 13 week treatment period or 4 week recovery period attributable to treatment.

BODY WEIGHT AND WEIGHT GAIN:
The overall bodyweight gain during the 13 week treatment period although variable was considered not to have been affected by treatment.
During the four week recovery period, the overall bodyweight gain was superior or similar to that of control among previously treated male and female groups.

FOOD CONSUMPTION:
The overall (Weeks 1 to 13) food consumption means for females were slightly, but statistically significantly, higher than control, although the mean values did not follow a dosage related trend and the difference from control was minor. The overall food consumption for males, were similar to control. During the four week recovery period, food consumption for female treated groups was comparable with control. Food consumption for males previously treated at 5 or 50 mg/kg/day were slightly higher than control, whilst at 15 mg/kg/day was comparable with control. These changes were considered to be fortuitous and not related to treatment.

FOOD EFFICIENCY:
Overall, food conversion efficiency ratios for all treated groups were considered to be essentially similar to control. During the recovery period, the efficiency for both sexes was variable, however, the overall ratios were superior to that of control among males and essentially similar to control among female recovery groups. The pattern of the overall ratios tended to follow a similar pattern to that of the recovery bodyweight gains.

OPHTHALMOSCOPIC EXAMINATION:
No effects were observed at the Week 12 examination.

HAEMATOLOGY:
Regarding the red blood cells, in Week 5, males at 50 mg/kg/day showed a slightly lower mean haematocrit (Hct) value and both sexes at this dose level, showed a higher reticulocyte mean value, when compared with controls. This differences were similar but greater in Week 13. In addition, both sexes at 50 mg/kg/day showed lower red blood cell values, with the males also showing a lower mean haemoglobin (Hb) value. Associated with these effects mean MCH and MCV values were slightly higher for both sexes at 50 mg/kg/day. There were no treatment-related effects recorded for the blood film comments. Recovery from the red blood cell effects was clearly evident by the end of the 4-week period without treatment. At the end of the recovery mean Hct values for both sexes and the mean Hb and RBC counts for females at 50 mg/kg bw/day, were slightly higher. This was considered to be due to an over compensation of red blood cell production following cessation of treatment, which was also reflected in the slightly lower than control mean reticulocyte value recorded for females at 50 mg/kg/day.
Regarding the wight blood cells, in Week 5 there were no clear effects of treatment on white blood cells. In Week 13, both sexes at 50 mg/kg/day and to lesser extent females at 15 mg/kg/day showed higher total white blood cell mean values. These differences were mainly due to higher lymphocyte counts in both sexes, with a doserelated trend apparent in females. A marginally higher neutrophil mean count was also noted for males at 50 mg/kg/day. Females at 15 or 50 mg/kg/day also showed higher than control monocyte and large unstained cell mean values. At the end of the recovery the counts at 15 mg/kg bw/day were similar to controls. Females at 50 mg/kg/day, continued to show a marginally higher mean lymphocyte and total white blood cell count, but the magnitude of the difference from control was lower, indicating recovery.

CLINICAL CHEMISTRY:
In both Weeks 5 and 13 males at 50 mg/kg/day showed higher mean bilirubin values. The magnitude of the difference from controls increased over time; however, full recovery was apparent after 4-weeks without treatment. In Week 5, males at 50 mg/kg bw/day and in Week 13 males and females at 15 and 50 mg/kg bw/day showed higher mean phosphorus values. Full recovery was evident after 4 weeks without treatment, but not for females treated at 50 mg/kg/day. Nevertheless, it was considered not to be of toxicological importance. Also in Week 13 only, females at 50 mg/kg/day showed higher mean cholesterol, total protein and albumin values. The higher than control values recorded in recovery were considered not to be clearly attributable to previous treatment. The other observed changed were not considered to be realated to treatment.

URINALYSIS:
In Week 13, males receiving 50 mg/kg/day showed a higher mean volume, whilst females showed a higher mean protein value. At the end of the recovery period volumes were similar to controls, although the mean protein value for females at this level was still higher than control. At the end of recovery the mean protein value for females at 5 mg/kg/day was identical to those treated at 50 mg/kg/day the results at 15 mg/kg/day were identical to controls, thus the higher mean protein value in the recovery period was considered not to be attributable to previous treatment. All other parameters are considered essentially similar to control.

NEUROBEHAVIOUR
Sensory reactivity: No effects were observed.
Grip strength: No effects were observed.
Motor activity: Males at 50 mg/kg/day showed increased low beam scores when compared with control at some time-points during the Week 5 investigation, with some mean values attaining statistical significance, however, total scores were within normal variation and no significant increases were observed at the Week 12 assessment of motor activity in either sex. Thus the effect owas considered to be fortuitous and not to be associated with treatment.

ORGAN WEIGHTS
In 13 weeks, kidney and spleen weights for males at 50 mg/kg bw/day were statistically significantly higher that control (adjusted for bodyweight). At the recovery, kidney weight were similar, while the spleen weights remained slightly higher, indicating partial recovery. Higher than control mean heart weights were recorded for females, however, the means did not follow a dosage related trend and no microscopic changes were seen in the heart, thus this change was not considered to be associated with treatment. After the 4 weeks recovery period, adrenal weights for males at 50 mg/kg/day were slightly higher than control. However, the mean values did not follow a dosage related trend. In addition adrenal weight at 50 mg/kg/day in Week 13 were marginally lower than control. Thus, in the absence microscopic changes, these differences are not considered to be associated with treatment. All other parameters are considered essentially similar to control.

GROSS PATHOLOGY
Macroscopic examination in Week 13, revealed enlargement of the spleen in males at 50 mg/kg/day when compared with control. At recovery no findings were observed and was considered not to be associated with treatment. The incidence and distribution of the other findings were considered to fall within the background range of macroscopic changes.

HISTOPATHOLOGY: NON-NEOPLASTIC
KIDNEY: Increased incidence and severity of cortical tubules with hyaline droplets, cortical tubular basophilia and medullary tubular basophilia were seen in males treated at 50 mg/kg/day. Increased incidence and severity of cortical tubular basophilia were also seen in males at 5 mg/kg/day, but not in males at 15 mg/kg/day. Tubular basophilia was unaffected in female rats. After recovery, no cortical tubules with hyaline droplets were present in any animal. Recovery
had occurred for cortical tubular basophilia or medullary tubular basophilia in all treated groups.
SPLEEN: Increased incidence and severity of extramedullary haemopoiesis was seen in all treated groups in both sexes. This was dosage related in males but not in females. Increased incidence and severity of haemosiderosis was seen in animals of both sexes at 50 mg/kg/day. After recovery extramedullary haemopoiesis and haemosiderosis had broadly resolved in both males and females, although moderate haemosiderosis was still evident in females at 50 mg/kg/day.
INCIDENTAL FINDINGS: All other microscopic findings were considered to be incidental and of no toxicological importance.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

(systemic toxicity)

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: (Basis for effect: effects on red blood cells, resulting in associated changes in the spleen)

2

Dose descriptor:

NOAEL

Remarks:

(reproductive organs)

Effect level:

> 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: (Basis for effect: no effects observed in reproductive organs or tissues)

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

No effects were observed in the reproductive organs at the highest dose tested (50 mg/kg bw/day): epididymides, ovaries, prostate, seminal vesicles, testes and uterus and cervix.

Applicant's summary and conclusion

Conclusions:

The 90day-NOAEL of the test substance in rats by oral route was 15 mg/kg bw/day in both sexes.
No effects were observed in the reproductive organs or tissues up to the highest dose tested (50 mg/kg bw/day).

Executive summary:

The systemic toxic potential of MIBKO was assessed when administered to rats by oral gavage over a period of 13 weeks followed by a 4 week recovery period. The test was performed according to OECD 408. 4 groups each comprising 10 rats per sex were exposed to 0 (control) 5, 15 or 50 mg/kg/day (2 mg/mL in corn oil). A further five male and five female rats were assigned to each of the groups. These animals were treated for 13 weeks, followed by a 4 week period without treatment to assess the recovery from any treatment related effect. During the study, clinical condition, detailed physical and arena observations, sensory assess recovery from any treatment related effect. reactivity, grip strength, motor activity, bodyweight, food consumption, ophthalmic examination, haematology, blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken (see the effects above, in the Details on results section). It was concluded that the principal action of MIBKO was to affect the red blood cell rate of turnover occurring mainly among animals receiving 50 mg/kg/day, resulting in associated changes in the spleen. Due to the higher incidence and severity of medullary haemopoesis observed in the spleen in both sexes among all treated groups, a No Observed Effect Level (NOEL) was not established for this study. A dosage level of 15 mg/kg/day, however, was considered to be the NOAEL for MIBKO in both sexes on this study, as the changes observed at 5 and 15 mg/kg/day were considered to be minor in nature and had shown full recovery after 4 weeks without treatment. No effects were observed in the reproductive organs or tissues up to the highest dose tested (50 mg/kg bw/day).