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

Carcinogenicity

Administrative data

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (limit test)

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 83-2 (Carcinogenicity)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japan/MAFF: Testing Guidelines for Toxicology Studies; Oncogenicity Study; p. 40 - 42. 1985

GLP compliance:

yes (incl. QA statement)

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 (used from May 23, 1991 to September 19, 1991), 91/180-1 (used from September 18, 1991 to April 29, 1992) and 91/180-2 (used from from April 23, 1992 to June 30, 1993); lab code letters: STUR)
- Lot/batch No.: N 27 (III a1), N 30 (III a2) and N 36 (III c1) respectively; date of manufaturing: April 05, 1991, June 17, 1991 and October 23, 1991 respectively
- Storage condition of test material: dark, +4°C
- Physical state: solid/whitish-brown powder
- Analytical purity: 94% and 95.9% respectively (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/female

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: the male animals were supplied on May 13, 1991 and the female animals on May 27, 1991 at an age of 32 days (males) or 33 days (females), respectively; 42 days old when the administration of the test substance started
- Weight at study initiation: animals of comparable weight; 192 g (172 - 215 g) for males and 149 g (132 - 170 g) for females at treatment begin.
- Fasting period before study: none specified
- Housing: singly in stainless steel wire mesh cages, 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: on the day of arrival a 10-day (males) or 9-day (females) adaption period started

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)

OTHER
At the end of the administration period, the animals were sacrificed after a fasting period (withdrawal of food) for at least 16-20 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): weekly
- Mixing appropriate amounts with (Type of food): for each concentration, the test substance was weighed out and mixed with a small amount of food in a beaker. Subsequently, a premix was prepared in a mixer by adding an appropriate amount of food followed by mixing. Then corresponding amounts of food, depending on dose group, were added to this premix in order to obtain the desired concentrations. 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 the diet over a period of 32 days and the homogeneous distribution was confirmed. The analytical results verified the correctness of the test substance concentrations in the diet.

Duration of treatment / exposure:

24 months

Frequency of treatment:

continuously in diet

Post exposure period:

None

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 9 , 36, 375, 770 mg/kg bw/day (in males)
Basis:

Doses / concentrations 2

Remarks:

Doses / Concentrations:
12, 47, 497 and 1046 mg/kg bw/day (in females)
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 3

Remarks:

Doses / Concentrations:
0, 200, 800, 8000 and 16000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

50

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: the selection of the doses for this study was based on the results of prior studies with the test substance:
1) In a 4-week study the test substance was administered to 5 male and 5 female Wistar rats per group at doses of 1000, 4000 and 16000 ppm via the diet. Following substance-induced findings were observed:
- 16000 ppm (about 1455 mg/kg body weight): (a) increased values of 1-glutamyltransferase and albumin in male animals
- 4000 ppm (about 370 mg/kg body weight and 1000 ppm (about 93 mg/kg body weight): (a) no substance-induced changes

2) In a 3-month study the test substance was administered to 10 male and 10 female Wistar rats per group at doses of 500, 2000, 8000 and 16000 ppm via the diet. Following substance-induced adverse findings were observed:
- 16000 ppm (about 1272 mg/kg body weight): (a) slightly reduced body weights in males, resulting in reduced values of about 6% at the end of the study; (b) reduced body weight change in males, resulting in reduced values of about 9% at the end of the study; (c) decrease in aspartate aminotransferase in the males; (d) increase in γ-glutamyltransferase in the serum of the males; (e) statistically significant increase of the relative liver weight was confirmed in both sexes; (f) distinct change of the distribution and the amount concerning the fatty infiltration in the liver of both sexes.
- 8000 ppm (about 625 mg/kg body weight): (a) slightly reduced body weights in males, resulting in reduced values of about 9% at the end of the study; (b) reduced body weight change in males, resulting in reduced values of about 14% at the end of the study; (c) decrease in aspartate aminotransferase in the males; (d) increase in γ-glutamyltransferase in the serum of the males; (e) statistically significant increase of the relative liver weight was found in the females; (f) distinct decrease of the amount and a change of the distribution pattern of the fat in the liver of the females.
- 2000 ppm (about 159 mg/kg body weight): (a) decrease in alanine aminotransferase and alkaline phosphatase in both sexes.
- 500 ppm (about 40 mg/kg body weight): (a) no substance-related adverse effects

In respect to the above mentioned findings, the following dose levels were chosen for the 24 month oncogenicity study: (1) 16000 ppm as highest dose level which should cause toxic effects. Moreover, this dose level should result in a test substance intake close to 1000 mg/kg body weight per day, which is the highest dose recommended for testing according to EPA's present MTD concept; (2) 8000 and 800 ppm: as intermediate doses; (3) 200 ppm: as lowest dose level which should result in a clear "no observed adverse effect level"

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
A check was made for dead or moribund animals twice a day (Mondays 0 Fridays) and once a day (Saturdays, Sundays and on Public holidays). If animals were in a moribund state, they were sacrificed and necropsied.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: examinations concerning evident signs of toxicity were carried out twice a day (Mondays 0 Fridays) and once a day (Saturdays, Sundays and on Public holidays). Additional comprehensive clinical examinations and palpations of the animals were carried out at least once a week until start of necropsy. Thereafter, the remaining animals were examined individually depending on the health status.
When body weight was determined, the clinical examinations were carried out on the day of weighing (e.g. day 0, day 7). Due to technical reasons, in the weeks without body weight determination these examinations were carried out not exactly at 7-day intervals, but on another day of the respective working week.

BODY WEIGHT: Yes
- Time schedule for examinations: body weight was determined before the start of the administration period in order to randomize the animals, then once a week during the first 13 weeks of the study, in each case on the same day of the week. After that, the body weight was determined at 4-week 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 AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: 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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: detailed clinical observations (see above) included the observation of lacrimation, exophthalmia and pupil size
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study (for animals in control and highest dose groups), or in all animals killed in extremis during the study.
- Anaesthetic used for blood collection: surviving animals were sacrificed by decapitation under CO anesthesia
- Animals fasted: Yes (only for the animals evaluated at the end of the study)
- How many animals: all
- Parameters examined: blood smears were prepared and evaluated

CLINICAL CHEMISTRY: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The surviving animals were sacrificed by decapitation under CO anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. The animals, which died intercurrently or were sacrificed in a moribund state, were necropsied as soon as possible after their death and assessed by gross pathology. The following weights were determined in all animals sacrificed on schedule: anesthetized animals, liver, kidneys, brain, adrenal glands, testes.

HISTOPATHOLOGY: Yes
The following organs or tissues were fixed in neutral buffered % formaldehyde solution: all gross lesions, adrenal glands, aorta, bone (sternum, femur), bone marrow (sternum), brain, cecum, colon, duodenum, epididymides, esophagus, eyes, female mammary gland area, femorotibial joint, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (mandibular, mesenteric), ovaries, oviducts, pancreas, parathyroid glands, pituitary gland, prostate gland, rectum, salivary glands (mandibular, sublingual), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid gland, trachea, urinary bladder, uterus, vagina, and all macroscopic abnormalities. Fixation was followed by histotechnical processing (hematoxylin-eosin), examination by light microscopy and assessment of findings.

Other examinations:

None

Statistics:

(1) Clinical examinations: mean and standard deviation were calculated for the variables feed consumption, body weight and test substance intake for each group and tabulated together with the individual values for feed consumption and body weight. The statistical significance of the clinical data (body weight) was checked using the KRUSKAL-WALLIS test and the MANN-WHITNEY U-test. (2) Hematology: mean and standard deviation were calculated for each test group and tabulated together with the individual values. Except for the differential blood count, a statistic one-way analysis of variance is done via the KRUSKAL-WALLIS test. If the resulting p-value is equal or less than 0.05 a pairwise comparison of each dose group with the control group was carried out. This comparison is done using the MANN-WHITNEY U-test for the hypotheses of equal medians. (3) Pathology: mean and standard deviation were calculated for the statistical evaluation of the study for the variables of body weight and of absolute and relative organ weights (related to body weight) of the animals in each test group and tabulated together with the individual values (absolute and relative organ weights). A non-parametric one-way analysis of variance was done via the KRUSKAL-WALLIS-h test. If the resulting p-value is less than 0.05 a pairwise comparison of each dose group with the control group was carried out. This comparison was performed using the WILCOXON-Test for the hypothesis of equal medians.

Results and discussion

Results of examinations

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):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
The mortality rate of males until day 735 was 30% in test group 0, 28% in test groups 1 and 2, 26% in test group 3 and 30% in test group 4. In females, the mortality rate until day 735 was 34% in test group 0, 30% in test group 1, 22% in test group 2, 26% in test group 3 and 22% in test group 4 (see Table 1). Thus, the mortality was assessed as being incidental and not related to treatment. Abnormal clinical signs were equally distributed between control and treated animals or occurred in single animals, only. Therefore, these findings were spontaneous in nature and do reflect the range of biological variation.

BODY WEIGHT AND WEIGHT GAIN
In males of test group 4, body weight was impaired statistically significant from day 7 onwards, resulting in reduced values of about 9% towards the end of the administration period. In males of test group 3, body weight was impaired statistically significant on day 7, from day 21 to day 567 and on day 735, resulting in reduced values of about 10% towards the end of the administration period. A similar impairment of body weight was seen in females of test groups 4 and 3, resulting in reduced values of about 14% (test group 4) and 13% (test group 3) towards the end of the administration period. No substance-related effects were seen in males and females of test group 1 and 2. The statistically significant deviations in females of test groups 1 and 2 on days 21 and/or 35 were assessed as being incidental.
Body weight change values reflected the impairment in body weight even more clearly: the values towards the end of the administration period were 12% (males) or 22% (females) below controls in test group 4 and about 13% (males) or 20% (females) below controls in test group 3.

FOOD CONSUMPTION
No substance-related effects were seen. All values were within the biological range of variation.

COMPOUND INTAKE (if feeding study)
The mean daily substance intake in mg/kg body weight for the entire administration period is represented in Table 2. In order to ascertain 4 weekly intervals, only the values of days 7, 35, 63, 91 and days 119 to 735 were taken into account for calculation of test substance intake.

FOOD EFFICIENCY
No changes suggesting clear influence on the food efficiency were seen when comparing the individual dose groups of either sex, with the corresponding control groups.

WATER CONSUMPTION
No substance-related overt changes in water consumption were observed.

HAEMATOLOGY
At termination of the study no substantial differences were seen in the results of the differential white blood cell count or in the red blood cell morphology in the control and highest dose groups (also of the prematurely sacrificed animals of both sexes). The marginal changes observed in isolated cases in animals treated with test substance (for white blood cell count) or both groups (for red blood cell morphology) are respectively not considered to be substance-related or seen in the red blood cells were noted both in the animals of the control groups and in the animals of the highest dose groups. These individual differences are incidental in nature or were caused by aging and cannot be associated with the treatment.

ORGAN WEIGHTS
- In the brain, an increase in relative weight was recorded for males of group 3 and females of groups 3 and 4, corresponding to the decreased terminal body weight.
- In the liver, an increase in relative weight by approx. 10% was recorded for females of group 4.
- In the kidneys, the absolute weight was decreased by approx. 7% in both sexes of group 3 and males of group 4.
- As compared with controls, the terminal body weights of males and females of groups 3 and 4 were significantly decreased. The group mean terminal body weighs of males of groups 3 and 4 were approx. 10%, those of females approx. 12 % and 14% lower than in controls
- With the exception of the decreased terminal body weighs and the increased relative liver weight, these differences were considered lo be toxicologically insignificant. The liver weight increase was mainly caused by the increased incidence of hepatic neoplasms.

GROSS PATHOLOGY
As compared with controls, the incidence of liver masses was remarkably increased in males and females of groups 3 and 4. In both sexes, liver masses occurred predominantly in animals that survived until the scheduled termination of the study. The incidence of cysts in the liver was remarkably higher in both sexes of group 4.

HISTOPATHOLOGY: NON-NEOPLASTIC
The incidence and degree of severity of eosinophilic foci of hepatocellular alteration was increased in males and females of group 4; minimal to severe bile duct proliferation increased in incidence and severity in females of test group 4.
A number of other histopathologic findings were recorded at a significantly different incidence or severity in treated groups, as compared with controls. They were either considered to be toxicologically insignificant (e.g. increased tubular mineralization in the kidneys of high dose males, increased ovarian cysts in groups 2 and 4) or to be of a spontaneous nature. Most of these lesions were age-associated and degenerative, inflammatory, or proliferative in character.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
In males and females of test groups 3 and 4, a significant increase in carcinomas of the liver was observed (see Table 3).
The statistically significant increase in liver carcinomas substantiated the necropsy findings of liver masses or cysts in males and females of groups 3 (8000 ppm) and 4 (16000 ppm). The incidence of carcinomas was higher in females than in males and was to be correlated with the increased number of foci of hepatocellular alteration. The majority of liver tumors was recorded in animals that were sacrificed at scheduled termination of the study. Of the limited number of decedents which had a liver tumor, only three were found with a probably or definitely fatal context of observation. None of the treated animals in dose groups 2, 3, and 4 died from a liver tumor. Of all liver carcinomas, three had metastasized into distant organs.
All other neoplastic and non-neoplastic findings recorded in this considered to be of a spontaneous nature. Their incidence and degree were similar in the control and treated groups.

Effect levels

Dose descriptor:

NOAEL

Effect level:

800 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

other:

Remarks:

Effect type: other: carcinogenicity and toxicity (migrated information)

Any other information on results incl. tables

Table 1: Cumulative mortality at the end of the study

Test groups	Concentration of test substance in the food (ppm)	Cumulative mortality (%)
		Male animals	Female animals
0	0	30	34
1	200	28	30
2	800	28	22
3	8000	26	26
4	16000	30	22

 

 

Table 2: Mean daily test substance intake in mg/kg bw fort he entire administration period

Test groups	Concentration of test substance in the food (ppm)	Mean daily substance intake (mg/kg bw)
		Male animals	Female animals
1	200	About 9	About 12
2	800	About 36	About 47
3	8000	About 375	About 497
4	16000	About 770	About 1046

 

Table 3: Liver tumour incidence

	Total animals with hepatocellular adenomas or carcinomas (animals with adenomas / animals with carcinomas) from a total of 50 evaluated animals per group (for a total of 100 animals per group)
	Group 0	Group 1	Group 2	Group 3	Group 4
Males	8 (1 / 7)	5 (0 / 5)	2 (0 / 2)	19* (1 / 18*)	11 (0 / 13)
Females	1 (0 / 1)	2 (1 / 1)	4 (2 / 2)	15* (2 / 13*)	17* (1 / 16*)
Total	9 (1 / 8)	7 (1 / 6)	6 (2 / 4)	34* (3 / 31*)	28* (1 / 27*)
*: significantly different from the control group

In conclusion, following substance-related findings were obtained in this study:

(1) 16000 ppm (about 770 mg/kg bw in males and 1046 mg/kg bw in females) dose group: (a) impairment in body weight, resulting in reduced values of about 9% in males and 14% in females towards the end of the administration period; (b) impairment in body weight change, resulting in reduced values of about 12% in males and 22% in females towards the end of the administration period; (c) increased relative liver weights in females; (d) increased incidence of liver carcinomas in females and in males and females combined (or in both sexes combined); (e) increase in incidence and degree of severity of eosinophilic foci of hepatocellular alteration in males and females; (f) increase in incidence and severity of bile duct proliferation in females.

 

(2) 8000 ppm (about 375 mg/kg bw in males and 497 mg/kg bw in females dose group: (a) impairment in body weight, resulting in reduced values of about 10% in males and 13% in females towards the end of the administration period; (b) impairment in body weight change, resulting in reduced values of about 13% in males and 20% in females towards the end of the administration period; (c) increased incidence of hepatocellular carcinomas in males and females and both sexes combined.

 

(3) 800 ppm (about 36 mg/kg bw in males and 47 mg/kg bw in females) dose group: (a) no substance-related findings.

 

(4) 200 ppm (about 9 mg/kg bw in males) and 12 mg/kg bw in females) dose group: (a) no substance-related findings.