kresoxim-methyl (ISO);methyl (E)-2-methoxyimino-[2-(o-tolyloxymethyl)phenyl]acetate

Specific investigations: other studies

Administrative data

Endpoint:

mechanistic studies

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Scientifically acceptable study (GLP, QA)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The objective of this study was to investigate the influence of the test substance on hepatic cell proliferation. In the 1-12-week study, rats were fed test substance doses equivalent to those used in a chronic bioassay and cell proliferation (S-Phase-Response; SPR) was determined using a 1-week continuous bromodeoxyuridine (BrdU)-labeling technique.

GLP compliance:

yes (incl. QA statement)

Type of method:

in vivo

Endpoint addressed:

carcinogenicity

Test material

Test material information

Constituent 1

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; test substance number: 91/180-2; lab code letters: STUR;
- Lot/batch No.: N 36 (III c1); Manufacturing date: October 23, 1991
- Storage condition of test material: dark, room temperature
- Physical state: beige powder
- Analytical purity: 92.7% (Reversed-Phase - HPLC with UV-Detection)
- Stability under test conditions: the storage stability was guaranteed over the study period
- Other: the homogeneity of the test substance was confirmed by analysis (Reversed-Phase - HPLC with UV-Detection)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Wistar rats (CHBB: Thom; SPF); from Dr. K. Thomae GmbH, D-W7950 Biberach/Riss, FRG
- Age at study initiation: 33 days old when supplied, 42 days old at start of the administration period
- Weight at study initiation: animals of comparable weight; 556.5 g (490 - 700 g) at treatment beginn.
- Fasting period before study: none specified
- Housing: singly in stainless steel wire, Type DK-III (Becker & Co., Castrop-Rauxel, FRG; floor area about 800 cm2)
- Diet (e.g. ad libitum): ground Kliba maintenance diet rat/mouse/hamster, 343 meal, supplied by Klingentalmuhle AG; CH-4303 Kaiseraugst, Switzerland
- Water (e.g. ad libitum): drinking water
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
The animals were housed in fully air-conditioned rooms. There were no deviations from these ranges which influenced the results of the study.
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12 (6:00 - 18:00 / 18:00 - 6:00 hours)

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): the test substance preparation was performed once before the beginning of the administration period
- Mixing appropriate amounts with (Type of food): the test substance was weighed and thoroughly mixed with a small amount of rodent feed in a beaker. Subsequently a premix was prepared in a household mixer by adding an appropriate amount of feed and mixing for approx. 3 min. Depending on the dose group, this premix was added to corresponding amounts of feed in order to obtain the desired concentrations, and mixing was carried out for about 10 minutes in a laboratory mixer.
- Storage temperature of food: room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in rodent feed at room temperature during at least 32 days and the homogeneity of the test substance preparation were confirmed analytically. Concentration controls were performed in samples drawn at the start of the administration period and about 10 weeks thereafter. The correctness of the concentration was proven (recovery rates of 97.0 - 101.9%)

Duration of treatment / exposure:

3 treatment duration were used: 1 week, 6 weeks and 13 weeks

Frequency of treatment:

continuously in diet

Post exposure period:

2 weeks in 1-week treatment period groups or 5 weeks in 13-week treatment groups (see Table 1); satellite groups were included in the 1 week and 13 weeks treatment duration groups

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 and 1658, 1662, 1362, 1140 and 1159 (in groups 1, 3, 5, 7 and 9; see Table 1) mg/kg bw per day
Basis:
other: Based on diet consumption; the content of test substance in the test substance/food mixes was determined by HPLC with UV detection.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0 and 16000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

5

Control animals:

yes, plain diet

Details on study design:

- Test groups and doses are summarized in Table 1; satellite groups were included for 1-week and 13-weeks treatment period.

- 1 week prior to necropsy, osmotic minipumps (see below) filled with BrdU (20 mg/ml in physiological saline) were implanted subcutaneously in the interlumbar region. Prior to implantation of the pumps, the rats were anesthetized by administering METOFANE®. At the end of the 3-week administration period all animals were sacrificed (no fasting period).

Examinations

Examinations:

MORTALITY: Yes
A check was made for dead and moribund animals twice Mondays to Fridays and once a day on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: a check was made twice Mondays to Fridays and once a day on Saturdays, Sundays and on public holidays for general observations. Once a week an additional comprehensive clinical examination was carried out.

BODY WEIGHT: Yes
- Time schedule for examinations: body weight was determined before the start of the administration period in order to randomize the animals. During the conduct of the study, the body weight was determined on day 0 (start of 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.

FOOD WATER CONSUMPTION: Yes
Food consumption was determined weekly over a period of 7 days. The values were calculated as grams per animal per day.

COMPOUND INTAKE (if feeding study): Yes

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

OTHER:
- Clinical Chemistry, Hematology and Urinalyses: blood, serum and urine parameters were not analyzed.

- Determination of Cell Proliferation
1) GROSS PATHOLOGY: Yes
The animals were sacrificed by decapitation under CO anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. The following weights were determined in all animals sacrificed on schedule: anesthetized animals and liver.

2) HISTOPATHOLOGY: Yes
The following organs or tissues were fixed in neutral buffered % formaldehyde solution: brain, pituitary gland, thyroid gland, parathyroid gland, thymus, trachea, lungs, heart, aorta, salivary glands (mandibular gland and sUblingual gland), liver, spleen, kidneys, adrenal glands, skin, pancreas, testes, accessory genital organs (epididymides, prostate, seminal vesicle, coagulation gland), esophagus, stomach (glandular and nonglandular stomach), duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, lymph nodes(Ln. mandibularis and Ln. mesenterialis), skeletal muscle, sciatic nerve, sternum (with bone marrow), bone marrow (femur), eyes, femur with knee joint, spinal cord (cervical, thoracal and lumbar cord). Fixation was followed by histotechnical processing (hematoxylin-eosin), examination by light microscopy and assessment of findings.

3) IMMUNOHISTOLOGY
Using the same block as in histopathology two slides of each specimen (liver and jejunum only) were prepared, one serving as the test slide, the other as a negative control slide. Staining was then done using Anti-Bromdesoxyuridin 1170 376 (Boehringer Mannheim GmbH, FRG) as primary antibody (Detection Kit: Super Sensitive Kit QA000-5L, BioGenex GmbH, FRG). The blocks were then incubated with chromogen substrate solution (Fast Red) and counterstained with hematoxylin.

4) QUANTITATIVE ASSESSMENT
From each liver lobe eleven photographs were taken (original magnification x 200). The localization of each photo was listed in a sketch, serving as guidance for a nearly regular distribution of measurement in all areas of the lobe. Each photograph shows comparable morphological structures. The periportal zone 1 (Rappaport) at the top, intermediate zone 2 and perivenous zone 3 following, depending on their extension.
Counts of immunopositive (red) and hematoxylinc ounter stained hepatocyte nuclei were made from all 11 fields (photos) from each lobe. Starting on the left side at the top approx. 100 nuclei were counted, representing zone 1. Subsequently another 100 nuclei representing zone 2 and partly zone 3 were counted. Imrnunopositive hepatic cells were counted separately. Nuclei of the bile duct epithelium, blood cells and mesenchymal cells were not counted. The labeling index (LI) in BrdU-immunostained sections was calculated as a ratio of labeled cells over labeled + unlabeled cells multiplied by 100.

Positive control:

none; jejunum serves as positive control tissue in immunohistological determination of cell proliferation and is therefore blocked together with liver.

Results and discussion

Details on results:

CLINICAL SIGNS AND MORTALITY
No substance-related findings were obtained.
No animal died during the course of the study.

BODY WEIGHT AND WEIGHT GAIN
In test group 7, body weight was statistically significantly impaired on several days (Table 2). This was assessed as being substance-related. No statistically significant deviations were seen concerning body weight change.

FOOD CONSUMPTION
In test group 7, food consumption was statistically significantly impaired on several days. In test group 9, food consumption was statistically significantly impaired on day 7 (Table 3). The findings in both groups were assessed as being substance-related.

COMPOUND INTAKE (if feeding study)
The approximate mean daily test substance intake (in mg/kg body weight per day) over the entire study period is shown in Table 4.

FOOD EFFICIENCY
No statistically significant deviations were seen.

ORGAN WEIGHTS
There was a statistically significant increase of the absolute and relative liver weights after one week of administration (Table 5).

GROSS PATHOLOGY
There were no morphological alterations related to the administration of the test substance.

HISTOPATHOLOGY
The microscopic findings noted are either single occurrences or they were recorded at biologically comparable incidences in the treatment groups and in the control group.

OTHER FINDINGS: Determination of Cell Proliferation (S-PhaseResponse)
- In group 1, there was a statistically significant increase in cell proliferation in zones 1 and 2 and a biologically significant increase in zone 3 (Table 6). The mean increase was 71% compared to the control animals. It was 75% in zone 1, 70% in zone 2 and 62% in zone 3.

- In group 3, there was a statistically significant decrease in cell proliferation in zones 1 and 2 and a biologically significant decrease in zone 3 (Table 6). The mean decrease was 57% compared to the control animals. It was a decrease of 60% in zone 1, 61% in zone 2 and 47% in zone 3.

- For group 5, there was a statistically significant increase in cell proliferation in total and in zone 1 (Table 6). The mean increase was 49% compared to the control animals, and it was 116% in zone 1. In zones 2 and 3 there was a slight, but not statistically significant decrease (8% in zone 2, 5% in zone 3).

- For group 7, there was a biologically significant increase in cell proliferation in total and in zones 1 and 2 (Table 6). The mean increase was 37% compared to the control animals. It was 44% in zone 1 and 39% in zone 2. In zone 3 there was a slight, but not statistically significant decrease of 7%.

- For group 9, there was a statistically significant decrease in cell proliferation in all three zones of the hepatic lobule (Table 6). The mean decrease was 70% compared to the control animals. The decrease was 67% in zone 1, 73% in zone 2, and 75% in zone 3.

- There was a statistically highly significant decrease in both recovery groups (groups 3 and 9) compared to the non-recovery groups (groups 1 and 7).

- After one week of administration the increase of the labeling index was similar in all three zones.

- After six weeks only zone 1 showed a strong reaction, whereas zones 2 and 3 exhibited a minimal decrease of the labeling indices compared to the control.

Any other information on results incl. tables

Following observation can be made based on the results:

 

1) Enhanced liver cell proliferation was observed in male rats after treatment for one, six or thirteen weeks. Compared to the controls (100%), the cell proliferation increase peaked after one week (171%; Table 6), an effect which is congruent with the increase of the absolute and relative liver weights (Table 5). After week six, the cell proliferation level was at 149% and after thirteen weeks at 137%, meaning that the test compound enhances DNA synthesis for a period of at least three months (Table 6).

 

2) Cell proliferation can be induced diffusely in all hepatocytes or it can be localized in a specific region of the lobule. Therefore, ten fields per lobe were evaluated, each field showing comparable morphological structures of the hepatic lobule. At an original 200-fold magnification the fields included zones 1, 2 and 3. In these fields approx. 100 cells were counted in each zone. The periportal area (zone 1) showed a pronounced increase in the labeling index, especially after six and thirteen weeks of administration. After one week cell replication was increased in all three zones to a similar extent. When treatment continued for 6 and 13 weeks, the effect diminished in zones 2 and 3. These data indicate a time-dependent selective effect of the test substance on the hepatocytes of zone 1.

 

3) The enhancement of cell replication, caused by the test compound administered is reversible. Compared to the controls, the values decreased to 43% after one week of administration and 2 weeks of recovery and to 30% after 13 weeks of administration and 5 weeks of recovery (Table 6). It is important to note that they remained below the control values in the sense of a counter regulation. The reduction of the DNA replication of 70% after five weeks of recovery supports the assumption that the sustained increase during the 13 weeks of treatment represents a key mechanism of the mode of action of the compound.

 

Table 2: Body weight (only groups 6 and 7; 13 weeks treatment; days 7, 21, 49, 56, 63, 70, 77 and 84, also showing no statistical differences are not included)

Group (dose)	Mean body weight (g) ± SD at indicated days after start of treatment
	Day 0	Day 14	Day 28	Day 35	Day 42	Day 91
6 (0 ppm)	187.3±4.8	286.3±6.9	346.8±13.0	375.1±12.2	394.6±15.9	495.9±22.8
7 (16000 ppm)	184.7±6.0	276.3±4.6*	329.6±11.8*	353.7±15.7*	368.7±17.3	461.4±29.3
*: significantly different from control value

 

Table 3: Food consumption (only groups 6 and 7; 13 weeks treatment; days 14, 21, 63 and 70, also showing no statistical differences are not included)

Group (dose)	Mean body weight (g) ± SD at indicated days after start of treatment
	Day 7	Day 28	Day 35	Day 42	Day 49	Day 56	Day 77	Day 84	Day 91
6 (0 ppm)	26.5±0.5	28.4±0.4	28.4±1.2	28.5±0.9	29.0±0.9	28.2±0.7	26.9±0.7	25.9±0.9	26.1±0.8
7 (16000 ppm)	23.8±0.7*	27.0±0.8*	23.3±1.1*	26.1±1.0*	26.8±1.3*	26.4±1.0*	25.2±1.0*	24.6±1.4	24.4±0.9*
*: significantly different from control value

 

Table 4: Test substance intake

Test group	Concentration in the diet (ppm)	Duration of treatment	Substance intake (mg/kg)
1	16000	1 week	1658
3	16000	1 week	1662
5	16000	6 weeks	1362
7	16000	13 weeks	1140
9	16000	13 weeks	1159

 

Table 5: Terminal body (BW) and liver (LW) weights

	Weight parameters for indicated treatment groups
	Group 0		Group 1		Group 2		Group 3		Group 4
	BW	LW	BW	LW	BW	LW	BW	LW	BW	LW
Ablsolute (g)	238.5±4.8	12.5±0.2	234.1±14.2	14.3±1.1	308.5±9.1	16.0±1.7	311.6±25.7	16.6±2.8	376.0±19.1	18.3±3.3
Relative (%)		5.2±0.13		6.2±0.12		5.2±0.4		5.3±0.5		4.9±0.6

 

Table 5 (continue): Terminal body (BW) and liver (LW) weights

	Weight parameters for indicated treatment groups
	Group 5		Group 6		Group 7		Group 8		Group 9
	BW	LW	BW	LW	BW	LW	BW	LW	BW	LW
Ablsolute (g)	275.2±14.3	20.2±2.0	488.8±22.2	21.8±2.6	454.8±28.0	19.6±1.0	528.4±64.3	22.3±4.2	484.6±38.3	22.1±4.1
Relative (%)		5.4±0.4		4.5±0.4		4.3±0.3		4.3±0.3		4.5±0.6

 

Table 6: Labeling index in the liver of the control animals and treated animals after 1-13 weeks of treatment. The table shows the absolute and relative values for the organ (total) and the single values for the three zones. 2000 hepatocytes were examined per zone, 6000 per animal; 5 animals per group.

 

 

Treatment Groups	Treatment period	Recovery period	Labeling index (LI)
			Total	%	Zone 1	%	Zone 2	%	Zone 3	%
0	1 week	-	14.90	100	16.86	100	19.12	100	8.73	100
1	1 week	-	22.48	171*	29.66	176*	32.57	170*	14.20	163
2	1 week	2 weeks	5.90	100	7.30	100	6.14	100	4.26	100
3	1 week	2 weeks	2.52	43*	2.89	40*	2.42	39*	2.25	53
4	6 weeks	-	2.48	100	3.41	100	2.44	100	1.60	100
5	6 weeks	-	3.70	149*	7.35	216*	2.24	92	1.52	95
6	13 weeks	-	1.31	100	2.58	100	0.82	100	0.55	100
7	13 weeks	-	1.79	137	3.72	144	1.14	139	0.51	93
8	13 weeks	5 weeks	1.56	100	2.68	100	1.42	100	0.57	100
9	13 weeks	5 weeks	0.47	30*	0.89	33*	0.39	27*	0.14	25*
*: significantly different from control value