methyl (2E)-{2-[(2,5-dimethylphenoxy)methyl]phenyl}(methoxyimino)ethanoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed in compliance with GLP regulations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): MEOE
- Physical state: solid, powder/light beige
- Analytical purity: 96.8g/100g
- Storage condition of test material: ambient (RT); no direct sunlight; avoid temperature above 40°C; protect against humidity

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Age at study initiation: 42 ± 1 days
- Weight at study initiation: Does not exceed +/- 20% of the mean weight of each sex
- Housing: The rats were housed together (5 animals per cage) in H-Temp polysulfonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2).
- Diet (e.g. ad libitum): Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland.
- Water (e.g. ad libitum): Food and drinking water (from water bottles) were available ad libitum.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: drinking water containing 1% carboxymethylcellulose

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
MEOE was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water containing 1% carboxymethylcellulose was filled up to the desired volume, subsequently released manually. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were produced once a week.

For details see table 1 below.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in drinking water containing 1% carboxymethylcellulose at room temperature for a period of 7 days was demonstrated before the start of the administration period.
Concentration control analyses of the test-substance preparations were performed in samples of all concentrations at the start of the administration period. Each 3 samples were taken from the lowest and highest and one sample from the mid concentration for homogeneity analysis; these samples were used as a concentration control at the same time.

Method: Quantitative H-NMR spectroscopy

Duration of treatment / exposure:

MEOE was administered daily for 28 days.

Frequency of treatment:

MEOE was administered daily for 28 days.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 1, 3 and 6 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
A check for moribund and dead rats was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If rats were in a moribund state, they were sacrificed and necropsied. All rats were checked daily before and within 2 hours as well as within 5 hours after the administration for any clinically abnormal signs. Abnormalities and changes were documented for each rat.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations (DCO) were performed in all rats prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The rats were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high). The following parameters were examined:
1. abnormal behavior during “handling”
2. fur
3. skin
4. posture
5. salivation
6. respiration
7. activity/arousal level
8. tremors
9. convulsions
10. abnormal movements
11. impairment of gait
12. lacrimation
13. palpebral closure
14. exophthalmus
15. feces (appearance/ consistency)
16. urine
17. pupil size

BODY WEIGHT: Yes
Body weight was determined before the start of the administration period in order to
randomize the rats. During the administration period the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

OPHTHALMOSCOPIC EXAMINATION: Yes / No / No data
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of study
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 per sex and dose
- Parameters checked: Leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Differential blood count, Reticulocytes, Prothrombin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of study
- Animals fasted: Yes
- How many animals: 5 per sex and dose
- Parameters checked: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, γ-Glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, triglycerides, cholesterol, magnesium, bile acids,

URINALYSIS: Yes
- Time schedule for collection of urine: day 23
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters checked: pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment, color, turbidity, volume

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

verious statistical tests

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
No rat died prematurely in the present study. Respiration sounds were observed in female No. 34 of test group 2 (3 mg/kg bw/d). This finding was observed from study day 21 onwards. This finding was assessed as being incidental and not related to the test substance.

BODY WEIGHT AND WEIGHT GAIN
No test substance-related changes of body weight and body weight gain were observed in any test group.
Body weight change was significantly increased in female animals of test group 1 (1 mg/kg bw/d) from study day 14 onwards (maximum +46.34% on study day 14). These changes were assessed as being incidental and not related to treatment.

When compared to control group 0 (set to 100%), the terminal body weight was significantly increased (+11%) in females of test group 1 (1 mg/kg bw/d). Because there was no doseresponse relationship the increase of the terminal body weight was regarded to be incidental. The mean absolute duodenum weight in test groups 1-3 (1, 3 and 6 mg/kg bw/d) did not show significant differences when compared to test group 0 (control).

FOOD CONSUMPTION
No test substance-related effects on food consumption were obtained.

WATER CONSUMPTION
No test substance-related findings were observed.

HAEMATOLOGY
No treatment-related, adverse changes among hematological parameters were observed.

In females of test group 3 (6 mg/kg bw/d) the absolute lymphocyte and, therefore, also the total white blood cell (WBC) counts were, although not significantly, higher compared to controls. The total white blood cell counts in the mentioned test group were within the historical control range whereas the absolute lymphocyte counts were slightly above the upper border of the historical control range (WBC: 3.06-5.03 Giga/L, absolute lymphocyte
counts: 2.66-3.85 G/L). Because these findings were single changes in clinical pathology of this test group, they were regarded as not adverse (ECETOC Technical Report No 85, 2002).

CLINICAL CHEMISTRY
No treatment-related, adverse changes among clinical chemistry parameters were observed.

In males of test group 1 (1 mg/kg bw/d) the serum calcium levels were lower and the glucose concentrations were higher compared to controls. Both parameters were not changed dosedependently and, therefore, this alteration was regarded as incidental and not treatmentrelated.

URINALYSIS
No treatment-related changes among urinalysis parameters were measured.

ORGAN WEIGHTS
The mean relative duodenum weight in test groups 1-3 (1, 3 and 6 mg/kg bw/d) did not show significant differences when compared to the control group 0.

GROSS PATHOLOGY
No treatment-related gross lesions were observed.

HISTOPATHOLOGY:
No histopathological findings were observed.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: highest dose tested

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Executive summary:

In conclusion the oral administration of MEOE by gavage over a period of 4 weeks revealed no signs of toxicity in male and female Wistar rats at dose levels up to 6 mg/kg bw/d. In addition, no increase of duodenum weight was observed at any dose level. Therefore, under the conditions of the present study, the no observed adverse effect level (NOAEL) was ≥6 mg/kg bw/d for male and for female Wistar rats.