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Diss Factsheets

Toxicological information

Neurotoxicity

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Administrative data

Endpoint:
neurotoxicity: acute oral
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP / Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1996

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EPA OPP 81-8 (Neurotoxicity Screening Battery)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
methyl (2E)-2-methoxyimino-2-[2-[(2-methylphenoxy)methyl]phenyl]acetate
EC Number:
604-351-6
Cas Number:
143390-89-0
Molecular formula:
C18 H19 N O4
IUPAC Name:
methyl (2E)-2-methoxyimino-2-[2-[(2-methylphenoxy)methyl]phenyl]acetate
Details on test material:
- Name of test material (as cited in study report): Reg. No. 242 009
- Physical state: powder / light brown
- Analytical purity: 92.7 %
- Stability under test conditions: was proven by re-analysis
- Lot/batch No.: N 36 = IIIc1

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Dr. Karl Thomae AG, Biberach, Germany
- Age at study initiation: 42 days
- Mean weight at study initiation: males: 190g; females: 147g
- Housing: Type DK III, stainless steel wire cages; floor area ~ 800 cm2; individual housing
- Diet (e.g. ad libitum): Kliba rats/mice/hamsters maintenance palleted diet; Klingenthalmuehle AG, Kaiseraugust, Switzerland; ad libitum (except FOB / motor activity test)
- Water (e.g. ad libitum): tap water; ad libitum (except FOB / motor activity test)
- Acclimation period: at least 9 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Vehicle: 0.5% aqueous carboxymethyl cellulose (CMC). Administration volume: 10 ml/kg bw.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
single administration
Frequency of treatment:
single administration
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 500, 1000, 2000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Examinations

Observations and clinical examinations performed and frequency:
Observations
The general state of health of the animals was checked twice daily on working days and once daily on weekends and public holidays. Furthermore, the animals were examined in detail and palpated once a week (see FOB examinations).

Body weight
The body weight of the animals was determined before the first FOB and motor activity measurement in order to randomise the animals (pre-exposure body weights on Day -6). Thereafter, body weights were determined on Day 0 (start of the administration period) and then 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.

Food consumption
Effects on food consumption were not examined.
Neurobehavioural examinations performed and frequency:
Functional observation battery (FOB) and Motor activity
A functional observational battery was performed with each animal on study on Day -6, on Day 0 (starting 2 hours after gavage), on Day 7 and Day 14. The FOB started with passive observations without disturbing the animals; followed by removal from the home cage, open field observations in a standard arena, sensorimotor tests and reflex tests as well as quantitative measurements. The findings were ranked according to the degree of severity, if applicable. The observations were performed blinded and in randomised order.
The parameters determined are given in the listing in table 1.

Motor activity was measured on the same day as the FOB. The measurement was performed in the dark using the Multi-Varimex System (Columbus Instruments Intl. Corp., Ohio, USA) with four infrared beams per cage. The measurements started at about 14:00 h (ca. 6 hours after gavage). The number of beam interrupts was counted over 12 intervals, each lasting five minutes. During the measurements the animals received no food and no water.
Sacrifice and (histo)pathology:
Sacrifice and neuropathology
At the end of the study period, 14 days after gavage administration, five animals per sex and test group were deeply anesthetized (Nembutal, ca. 4 ml/kg bw) and sacrificed by perfusion fixation. The sacrificed animals were necropsied and the visible organs assessed by gross pathology as thoroughly as possible for perfused animals. Soerensen's phosphate buffer served as rinsing solution and Karnovsky was used as fixation solution.

With the exception of peripheral nerve specimen, all the tissues listed in the table below from perfused animals of the control and high-dose group were embedded in Paraplast (paraffin).

See table 2 for tissues examined.

Peripheral nerve samples from all 5 of the perfused control group and high-dose group animals from each sex were embedded in epoxiresin-based plastic. Semi-thin cross and longitudinal sections were prepared and stained with toluidine blue before light-microscopical examination. Fixed peripheral nerve specimens from the low- and mid-dose group were stored in buffer solution but not further processed or evaluated.

Cross and/or oblique sections were prepared and stained with hematoxylin/eosin (H&E), followed by assessment of the tissue sections by light microscopy. Tissue samples from perfused rats of the low- and mid-dose group were preserved in neutral buffered 4% formaldehyde solution but not further processed or evaluated.

All animals not subjected to perfusion fixation were sacrificed under CO2 anesthesia without further examinations.
Positive control:
Positive control data:
The examinations were carried out by trained technicians which performed positive control studies as part of their training. Acrylamide, Trimethyltin chloride, 3,3'-Iminodiproprionitril, Carbaryl, Nomifensin and Diazepam were used as positive control compounds. In the positive control studies, behavioral and neuropathological sequelae of substances with nervous system effects were evaluated using Functional Observational Batteries (FOB), Motor Activity Measurements and Neuropathology. Clinical signs of peripheral neuropathy (e.g. ataxia, limb weakness), central neuropathy (e.g. tremors) and autonomic signs (e.g. salivation) could be shown. Histopathologically, changes in the peripheral nervous system (e.g. Wallerian-like degeneration) and central nervous system (e.g. neuronal necrosis) were seen. The motor activity device could show both increased and decreased activity. The interobserver reliability of the technicians performing functional observational batteries was proven. Summaries of the positive control studies are presented in the study report
Statistics:
Body weight: Parametric one-way analysis of variance via the (2-sided) F-test (ANOVA). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using the DUNNETT's test (2-sided) for the hypothesis of equal means.
Quantitative FOB parameters and motor activity: Non-parametric one-way analysis using the KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using MANN-WHITNEY U-test (two sided) for the equal medians

Results and discussion

Results of examinations

Details on results:
Clinical signs of toxicity: No abnormal clinical signs were detected.

BODY WEIGHT
No statistically significant or biologically relevant deviation from body weight control group values was observed development was not impaired in males or females at any dose level (see table 1).

Mortality: No deaths occurred during the study.

Functional Observational Battery (FOB):
- Home cage observations: No substance-related effects were observed.
- Open field observations: No substance-related effects were observed.
- Sensormotor tests / reflexes: Grip strength of forelimbs was decreased statistically significant in high and mid dose females on Day 14. However, as the values were very close to the control values, a biologically significant effects is not very plausible. Moreover, all values were well within the historical range. Thus, these findings were assessed as being only statistically, but not biologically relevant. A substance-related effect can therefore be excluded.

No statistically significant deviations were obtained in any of the other dose groups. In summary, no substance-related findings were observed in any part of the functional observation battery.

Motor Activity Measurement
Regarding the overall motor activity the values were statistically significantly increased in high and mid dose males on day -6, and in high dose males on day 14. However, as there was no dose-response relationship and as the changes were observed even prior to treatment, this was assessed as being incidental.

Comparing the single intervals with the control groups several values of the treatment groups (both sexes) differed statistically significantly from control values. However, as there was no dose-response relationship and the changes were observed even prior to treatment, these deviations were assessed as being incidental.



NECROPSY AND NEUROPATHOLOGY

Gross lesions: There were no gross lesions noted.

Light microscopy (neuropathology): One female test animal of the high dose (Animal-No. 73) showed a minimal axonal degeneration of the proximal sciatic nerve, which is seen to be a spontaneous lesion for reasons as follows:
- No further high dose test animals are concerned
- no further nerve fiber samples in this test animal are concerned,
- the lesion is not a specific neurotoxic effect and it is minimal in grading.

The light-microscopic investigation did not reveal any substance-dependent changes in the organ samples examined. Especially, no substance-induced neuropathological lesions could be detected.

Effect levels

Dose descriptor:
NOAEL
Effect level:
2 000 other: mg/kg bw
Sex:
male/female
Remarks on result:
other:

Any other information on results incl. tables

 

Males

Females

Dose level [mg/kg bw]

0

500

1000

2000

0

500

1000

2000

Body weight [g]

    Day -6                    [g]

                                    (%)

134.2

(100)

134.2

(100)

133.7

(99.6)

134.4

(100.2)

115.7

(100)

115.3

(99.6)

114.5

(98.9)

115.2

(99.6)

     Day 0                    [g]

                                    (%)

189.2

(100)

185.1

(97.8)

195.1

(103.1)

189.3

(100.1)

148.0

(100)

147.1

(99.4)

146.4

(98.9)

147.7

(99.8)

     Day 7                    [g]

                                    (%)

235.9

(100)

227.5

(96.4)

246.1

(104.3)

238.4

(101.0)

169.1

(100)

169.1

(100)

169.4

(100.2)

172.8

(102.2)

   Day 14                    [g]

                                    (%)

280.3

(100)

266.0

(94.9)

292.2

(104.2)

283.5

(101.2)

188.0

(100)

186.0

(101.0)

189.9

(101.0)

189.7

(100.9)

Statistical evaluation: * p=< 0.05, ** p =< 0.01 (Dunnett's test, 2-sided)

Table 1: Body weight.

Historical control data - Grip strength forelimbs

Age:

6 weeks

7 weeks

8 weeks

FIRST MEASUREMENT (Newton)

Males

2.8 (1.5 – 4.2)

3.3 (1.4 – 5.0)

3.8 (1.6 – 5.2)

Females

2.8 (1.4 – 4.2)

3.3 (1.8 – 4.8)

3.3 (2.0 – 5.0)

SECOND MEASUREMENT (Newton)

Males

2.9 (1.8 – 4.5)

3.3 (1.4 – 5.3)

3.7 (1.8 – 5.8)

Females

2.9 (1.8 – 4.2)

3.4 (1.8 – 5.0)

3.4 (1.8 – 5.6)

Data is given as mean (Newton) and minimum-maximum range (in brackets)

19 studies with Wistar rats from the BASF test facility

 

Males

Females

Dose level [mg/kg bw]

0

500

1000

2000

0

500

1000

2000

Grip strength forelimbs [Newton]

  Day -6               [N]

                           (%)

2.7

(100)

2.3

(82.7)

2.7

(99.6)

2.8

(100.7)

3.4

(100)

3.1

(90.9)

2.8

(83.5)

3.2

(95.0)

   Day 0               [N]

                           (%)

3.2

(100)

3.0

(92.5)

3.4

(106.3)

3.3

(104.1)

3.4

(100)

3.3

(95.9)

3.5

(103.2)

3.4

(99.7)

   Day 7               [N]

                           (%)

3.8

(100)

3.8

(101.1)

3.9

(101.9)

3.8

(101.1)

3.3

(100)

3.2

(96.8)

3.2

(95.8)

3.1

(92.4)

 Day 14               [N]

                           (%)

3.9

(100)

3.6

(91.4)

3.9

(99.0)

3.8

(95.9)

3.4

(100)

3.3

(98.2)

3.0*

(87.6)

2.9**

(86.7)

Statistical evaluation: * p£ 0.05, ** p£ 0.02; *** p£ 0.002 (Kruskal-Wallis, 2-sided)

Table 2: Grip strength forelimbs

Applicant's summary and conclusion