Teflubenzuron

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 January 1984 to 06 May 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Remarks:

DRF including analysis of stability of test subtance in feed

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

18 April 1983 to 16 May 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

No histopathological examination.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: Wistar KFM-Han.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: KFM Kleintierfarm Madoerin AG in 4414 Fuellinsdorf/Switzerland
- Age at study initiation: 5 weeks
- Weight at delivery (4 weeks): 41 to 55 g for males and 43 to 56 g for females.
- Housing: in groups of five in Makrolon cages type-4 with wire mesh top
- Diet: pelleted standard Kliba no. 343 rat/mouse maintenance diet.
- Water: ad libitum, tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: April 18, 1983 To: May 16, 1983

Route of administration:

oral: feed

Details on oral exposure:

DIET PREPARATION
The test substance was mixed with the microgranulated feed. Water was added to the feed preparations at 1:10 volume/weight ratio to ensure pelleting. The preparations were repelleted with a Buehler pelleting machine, Type DFP (Buehler AG, Uzwil/Switzerland). The pellets were air-dried for 24 hours and then stored at room temperature in paper bags.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

After mixing of the diet, samples were taken from the top (T), middle (M) and bottom (B) of the batches. The samples were either immediately frozen and kept at -20° C until analysis.
Extracts were prepared from feed and pre-analysis clean-up was used (for fatty residues and by column chromatography) as preparation. The content of active compound in the extraction solutions was determined directly by high performance liquid chromatography (HPLC).
Recovery experiments carried out routinely throughout the course of the feeding study series gave recovery values of 94 to 101 % (100 ppm applied).

Duration of treatment / exposure:

28 days

Frequency of treatment:

continuous

Dose / conc.:

0 ppm

Dose / conc.:

100 ppm

Remarks:

Males 12.8 mg/kg bw/ day, Females 11.7 mg/kg bw/ day
Mean calculated food nominal dosage level (from Day 3, 10, 17 and 24).

Dose / conc.:

1 000 ppm

Remarks:

Males 133.6 mg/kg bw/ day, Females 113.8 mg/kg bw/ day
Mean calculated food nominal dosage level (from Day 3, 10, 17 and 24).

Dose / conc.:

10 000 ppm

Remarks:

Males 1254.2 mg/kg bw/ day, Females 1219.2 mg/kg bw/ day
Mean calculated food nominal dosage level (from Day 3, 10, 17 and 24).

No. of animals per sex per dose:

5

Control animals:

yes, plain diet

Details on study design:

- Fasting period before blood sampling for clinical biochemistry: overnight

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on weekdays and once daily on weekends.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily on weekdays and once daily on weekends.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Food consumption was recorded over a continuous measuring period of 7 days on a Mettler PK 4800 balance. The mean daily food consumption was calculated and reported weekly.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 weeks of treatment.
- Anaesthetic used for blood collection: Yes (light ether anesthesia)
- Animals fasted: Yes (overnight)
- How many animals: all (5 animals/sex/group)
- EDTA-2Na was used as anticoagulant during blood collection (hematology).
- Na-Citrate, 3.8% was used as anticoagulant during blood collection (coagulation).
The following commercial reference controls were used to monitor the performance of the method:
Hematology: Control blood - level I (low range), Control blood - level II (normal range), Control blood - level III (high range) (Merz & Dade AG, Duedingen/Switzerland).
IL 282 Co-Oximeter Control (Instrumentation Laboratory Inc., Lexington, Ma./USA).
Coagulation: Ci-Trol-1 (normal range), Ci-Trol-2 (high range) (Merz & Dade AG, Duedingen/Switzerland).
The following parameters were evaluated: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Reticulocyte count, Nucleated erythrocytes- normoblasts, Heinz bodies, Methemoglobin, Total leukocyte count, Differential leukocyte count, Red cell morphology.
Coagulation: Thromboplastin time, Partial thromboplastin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 weeks of treatment.
- Animals fasted: Yes (overnight)
- How many animals: all (5 animals/sex/group)
Li-heparin (140 U.S.P. Units) was used as anticoagulant during blood collection:
The following commercial reference controls were used to monitor the performance of the method:
Clinical Biochemistry: Seronorm (normal range), Pathonorm L (low range), Pathonorm H (high range)(Nyegaard & Co., Oslo/Norway).
Moni-Trol I-E (normal range), Moni-Trol II-E (abnormal range) (Merz & Dade AG, Duedingen/Switzerland).
Protein electrophoresis: Seronorm Protein (Nyegaard & Co., Oslo/Norway).
The following parameters were determined:
glucose, urea, creatinine, bilirubin total, cholesterol total, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, sodium, potassium, chloride, protein total, protein electrophoresis.

URINALYSIS: Yes
- Time schedule for collection of urine: after 4 weeks of treatment.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (overnight)
Urine samples were collected by isolation in special metabolism cages overnight (18 hours), using clean brown specimen battles. During this time the animals were deprived of food, but not of water. The following commercial reference controls were used to monitor the performance of the method:
Urinalysis: Clini-Tek performance capsules, Tek-Chek 4 (Ames Division, Miles Laboratories, Inc., Elkhart, Indiana/USA).
The following listed parameters were determined: Volume (18 h), specific gravity, pH, protein, glucose, ketones, bilirubin, blood, nitrite, urobilinogen, urine sediment.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Necropsy was performed on all rats. The animals were anesthetized by intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination.

HISTOPATHOLOGY: No

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related signs or symptoms were observed.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weights of the animals of groups 2, 3 and 4 were slightly lower than those of the control animals at pretest week 1. Despite the initial differences, the mean body weight gain of all treated male and female rats was comparable to that of the control animals.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean food consumption of groups 1, 2 and 3 and of the females of group 4 was similar.
The mean food consumption of the group 4 males was slightly reduced during the entire treatment period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematology data showed no differences among groups.
Treatment-unrelated:
All statistical differences in the results of the hematologic parameters were considered incidental and of normal biological variation.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related:
A slight, yet dose-dependent increase in the enzyme activities of plasma transaminases (ASAT and ALAT) was observed in the animals of the mid- and high-dose groups.
Treatment-unrelated:
All other statistical differences in the results of the parameters were considered incidental and of normal biological variation.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Urinalysis data showed no differences among groups.
Treatment-unrelated:
All differences in the results of the urinalysis parameters were considered incidental and of normal biological varation.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No grass lesions were encountered in any of the animals in all groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Dose descriptor:

NOAEL

Remarks on result:

not determinable because of methodological limitations

Remarks:

The design of the study, because of the lack of histological examination, is not suitable to establish a NOAEL.

Mean daily substance intake:

Males
	group
	0 ppm	100 ppm	1000 ppm	10000 ppm
Day (week)	Mean daily substance intake (mg/kg bw/d)
3 (1)	0	15.64	160.90	1599.88
10 (2)	0	13.67	145.17	1346.23
17 (3)	0	12.11	124.10	1121.32
24 (4)	0	9.78	104.32	949.33
Mean	0	12.8	133.62	1254.19
Females
	group
	0 ppm	100 ppm	1000 ppm	10000 ppm
Day (week)	Mean daily substance intake (mg/kg bw/d)
3 (1)	0	13.55	138.63	1415.76
10 (2)	0	11.93	117.99	1254.11
17 (3)	0	11.4	105.62	1184.46
24 (4)	0	9.85	92.82	1022.58
Mean	0	11.68	113.77	1219.23

 

 

Selected parameters of clinical biochemistry:

Parameter	group
	0 ppm	100 ppm	1000 ppm	10000 ppm
Males
ASAT / GOT [ukat/L]	1.30	1.34	2.02**	2.34**
ALAT / GPT [ukat/L]	0.52	0.55	1.10**	1.49**
Females
ASAT / GOT [ukat/L]	1.18	1.36	3.14	4.23**
ALAT / GPT [ukat/L]	0.37	0.46	1.51	2.13*

* T-Test based on pooled variance significant at 5% level

** T-Test based on pooled variance significant at 1% level

Cross-reference 3

Reason / purpose for cross-reference:

reference to other study

Remarks:

DRF

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

06 July 1983 to 01 November 1983

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Remarks:

contains analysis of stability of test item in feed

Qualifier:

according to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

Version / remarks:

22 August 1978, revised in 1982

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Wistar KFM-Han.

Details on species / strain selection:

This test system has been internationally recommended as a suitable rodent species for assessing the potential toxic effects of the test substance.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: KFM Kleintierfarm Madoerin AG, 4414 Fuellinsdorf/Switzerland
- Age at study initiation: 5 weeks
- Initial weight (pretest week 1): males 62 - 80 g, females 60 - 73 g
- Housing: in groups of 5 in Makrolon type-4 cages with wire mesh tops
- Diet: ad libitum, pelleted standard Kliba 343 rat/mouse maintenance diet (Klingentalmuehle AG, 4303 Kaiseraugst/Switzerland).
- Water: ad libitum, tap water
- Acclimation period: 1 week

DETAILS OF FOOD AND WATER QUALITY:
Certificates for the analysis of contaminants of the batches were attached to the study report.
Certificates for the analyses (chemical and bacteriological) were attached to the study report.
The food and water quality was considered to be acceptable.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: July 06, 1983 To: November 01, 1983

Route of administration:

oral: feed

Details on oral exposure:

DIET PREPARATION
The test substance was mixed with microgranulated feed using a mixer (Buehler, type DDMA-0.5, Buehler AG, Uzwil/Switzerland) and compressed with a pelleting machine (Buehler, type DFPL, Buehler AG, Uzwil/Switzerland). Water was added to each feed preparation at a 1:10 volume/weight ratio to ensure pelleting, after which they were air-dried. The feed preparations were stored in disposable paper bags at 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 feed was determined during the range-finding study (see cross-referenced study).
At monthly intervals, feed samples from all groups were analyzed for concentration and homogeneity by the Analytic Chemistry Laboratory of CELAMERCK GMBH & CO., KG.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

continuous

Dose / conc.:

0 ppm

Dose / conc.:

100 ppm

Remarks:

100 ppm (equivalent to 8.02 mg/kg/day in males and 9.12 mg/kg/day in females)
Mean calculated food nominal dosage level.

Dose / conc.:

1 000 ppm

Remarks:

1000 ppm (equivalent to 81.56 mg/kg/day in males and 94.01 mg/kg/day in females)
Mean calculated food nominal dosage level.

Dose / conc.:

10 000 ppm

Remarks:

10000 ppm (equivalent to 809.02 mg/kg/day in males and 942.43 mg/kg/day in females)
Mean calculated food nominal dosage level.

No. of animals per sex per dose:

10 for 13-week exposure (group 1 to 4)
5 for 13-week exposure with subsequent 4-week recovery (group 1 and 4)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were based upon data from a 4-week range-finding study (see cross-referenced IUCLID entry).
- Fasting period before blood sampling for clinical biochemistry: overnight (18-hour)
- Satellite groups: after 13 weeks treatment, additional 5 animals per sex and dose (control and high dose only) were kept for a four week recovery period.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was examined for changes in appearance and behavior daily, and a detailed weekly clinical examination, which included palpation for tissue masses.

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly

FOOD CONSUMPTION:
- over a 7-day period using an electronic recording system (see body weights). The daily food consumption was calculated and reported in weekly intervals.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to dosing and at 13 and 17 weeks.
- Dose groups that were examined: 13-week application groups and recovery groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 6 and 13 weeks of treatment, and from all animals of the recovery group prior to sacrifice.
- Anaesthetic used for blood collection: Yes (light ether anesthesia).
- Animals fasted: Yes (18 hours)
- How many animals: all (10/sex/group 13-week application, 5/sex of Group 1 and 4 (Recovery))
- EDTA-2Na was used as anticoagulant during blood collection (hematology).
- Na-Citrate, 3.8% was used as anticoagulant during blood collection (coagulation, 1:10).
The following commercial reference controls were used to monitor the performance of the method:
Hematology: Control blood - level I (low range), Control blood - level II (normal range), Control blood - level III (high range) (Merz & Dade AG, Duedingen/Switzerland).
IL 282 CO-Oximeter Control (Instrumentation Laboratory Inc., Lexington, Ma./USA).
Coagulation: Ci-Trol-1 (normal range), Ci-Trol-2 (high range) (Merz & Dade AG, Duedingen/Switzerland).
The following parameters were investigated: erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, reticulocyte count, nucleated erythrocytes- normoblasts, Heinz bodies, methemoglobin, total leukocyte count, differential leukocyte count, red cell morphology.
Coagulation: Thromboplastin time, partial thromboplastin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 6 and 13 weeks of treatment, and from all animals of the recovery group prior to sacrifice.
- Animals fasted: Yes (18 hours)
- How many animals: all (10/sex/group 13-week application, 5/sex of Group 1 and 4 (Recovery))
Li-heparin (140 U.S.P. Units) was used as anticoagulant during blood collection:
The following commercial reference controls were used to monitor the performance of the method:
Clinical Biochemistry: Seronorm (normal range), Pathonorm L (low range), Pathonorm H (high range)(Nyegaard & Co., Oslo/Norway).
Moni-Trol I-E (normal range), Moni-Trol II-E (abnormal range) (Merz & Dade AG, Duedingen/Switzerland).
Protein electrophoresis: Seronorm Protein (Nyegaard & Co., Oslo/Norway).
The following parameters were determined:
glucose, urea, creatinine, total bilirubin, total cholesterol, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, gamma-glutamyltransferase, ornithine carbamyltransferase, calcium, phosphorus, sodium, potassium, chloride, total protein.

URINALYSIS: Yes
- Time schedule for collection of urine: after 6 and 13 weeks of treatment, and from all animals of the recovery group prior to sacrifice.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
Urine samples were collected by metabolism cage into clean brown specimen bottles. The following commercial reference controls were used to monitor the performance of the method:
Urinalysis: Chek-Stix Urinalysis Control Strips (Ames Division, Miles Laboratories, Inc., Elkhart, Indiana/USA).
The following listed parameters were determined: Volume (18 h), specific gravity, pH, protein, glucose, ketone, bilirubin, blood, nitrite, urobilinogen, urine sediment.

OTHER:
Organ weights
The following organ weights were determined for all rats: brain, heart, liver, kidneys, lungs, spleen, thymus, adrenal glands, thyroid gland, pituitary gland, testes/ovaries.

Sacrifice and pathology:

The animals were anesthetized by intraperitoneal injection of Pentobarbital (2 mL/kg body weight) and sacrificed by exsanguination.
Necropsy was performed on all rats. At postmortem examination, all organs were inspected; all findings were recorded.

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Tissues were fixed in 4% neutral formalin and embedded in Paraplast. The sections were cut at a nominal thickness of 4 µm and stained with hematoxylin and eosin.
Sections of the following organs and tissues of all control and high-dose rats were examined microscopically:
Brain (cross-sections of cerebrum, cerebellum and pans, and medulla oblangata), spinal cord, sciatic nerve, heart, aorta, trachea, lungs, tongue, esophagus, stomach, duodenum, jejunum, Ileum, cecum, colon, rectum, liver, pancreas, kidneys, urinary bladder, testes, epididymides, prostate, seminal vesicles, ovaries, uterus, pituitary gland, thyroid gland, parathyroid glands, adrenal glands, spleen, bone marrow, thymus, mandibular lymph node, mesenteric lymph node, salivary glands, mammary glands, skin and appendages, skeletal muscle, bone (sternum), eyes with optic nerve, Harderian glands, gross lesions

From the low- and mid-dose rats, only heart, liver, kidneys, lungs, and gross lesions were examined microscopically.

Statistics:

The following statistical methods were used to analyze body weights, food consumption, organ weights and clinical laboratory data:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences if the variables could be assumed to follow a normal distribution.
Student‘s T-test, based on a pooled variance estimate, was used for intergroup comparisons (i.e. single treatment groups against the control group).
William's test was used to determine the lowest dose group significantly different from the control group, if a monotone (increasing or decreasing) dose-response relationship existed.
The difference of the overall mean of the treated groups to the control group was tested for significance if some evidence existed for differences between the treatment groups and the control group, and a monotone dose-response relationship and significant T-tests were absent.
The median test was used to assess the significance of intergroup differences if the variables were defined on a discrete scale.
Fisher‘s exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.
The adjustment for multiple testing was performed by comparing the expected and observed number of significant results obtained.
Individual values, means, standard deviations and t-statistics were rounded off before printing. T-tests were calculated on the basis of exact values for means and pooled variances and rounded off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different T-statistic values with respect to the control group.

References:
Williams, D.A. (1971/72). Biometrics 27: 103-117 and 28: 519—531.
Fisher, R.A, "Statistical Methods for Research Workers", Oliver and Boyd, Edinburgh (1950).

Clinical signs:

no effects observed

Description (incidence and severity):

No signs of toxicity and no symptoms which could be related to the treatment were observed.

Mortality:

no mortality observed

Description (incidence):

No deaths occurred during the course of this study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no differences of body weight gain between treated animals and controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean food consumption of all animals was similar.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment-related findings were recorded.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The assessment of hematological data revealed no treatment-related changes.
Treatment-unrelated:
All statistical differences in the results of the hematological parameters were considered incidental and of normal biological variation.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Compared to control rats, the following statistically significant changes were noted after 6 and 13 weeks of treatment:
- slightly increased aspartate aminotransferase (ASAT) activity for male and female rats of groups 3 and 4 after 6 weeks and moderately increased ASAT activity for male rats of group 4 after 13 weeks,
- slightly increased alanine aminotransferase (ALAT) activity for male rats of groups 3 and 4 after 6 weeks and moderately increased ALAT activity for male rats of group 4 after 13 weeks,
- slightly increased alkaline phosphatase (ALP) activity for male rats of group 4 after 6 and 13 weeks,
- slightly increased ornithine carbamyl transferase (OCT) activity for male rats of group 4 after 6 weeks, moderately increased OCT activity for male rats of group 4 after 13 weeks, and slightly increased OCT activity for female rats of group 4 after 13 weeks.

No such differences among groups were noted at the end of the recovery period.
The possible toxicological significance of these differences in the subchronic treatment was not supported by any pathological observation. However, the small increases of enzyme activities may be considered to be of biological significance.

Treatment-unrelated:
All other statistical differences in the results of the biochemical parameters were considered incidental and of normal biological variation.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

The assessment of urinalysis data revealed no treatment-related changes.
Treatment-unrelated:
All differences in the results of the urinalysis parameters were considered incidental and of normal biological variation.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The organ weight characteristics showed significantly enlarged livers in group 4 females after 13 weeks (p < 0.01). In group 4 males, evidence of enlarged testes after 13 weeks was noted when compared with the values of the control group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A number of minor lesions were encountered at a random incidence in various organs of control and treated rats. These were common findings usually encountered in rats of this strain and age.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

A number of spontaneous lesions were randomly observed in various organs of control and treated rats. Type, incidence, and severity of these lesions were similar in all groups.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

A number of spontaneous lesions were randomly observed in various organs of control and treated rats. Type, incidence, and severity of these lesions were similar in all groups.

Key result

Dose descriptor:

NOAEL

Effect level:

81.56 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical biochemistry

Remarks on result:

other: 1000 ppm equivalent to 81.56 mg/kg bw/day in males

Key result

Dose descriptor:

NOAEL

Effect level:

94.01 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical biochemistry

organ weights and organ / body weight ratios

Remarks on result:

other: 1000 ppm equivalent to 94.01 mg/kg bw/day in females

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

10 000 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Test substance intake:

	Mean test substance intake [mg/kg bw/d]
Nominal concentration in feed [ppm]	Males	Females
100	8.02	9.12
1000	81.56	94.01
10000	809.02	942.43

 

Selected clinical chemistry parameters:

Nominal concentration in feed [ppm]	N	ASAT [µkat/L]	ALAT [µkat/L]	ALP [µkat/L]	OCT [nkat/L]
		Males, 6 weeks
0	15	1.30 ± 0.13	0.53 ± 0.11	3.26 ± 0.41	40.72 ± 20.19
100	10	1.36 ± 0.11	0.71 ± 0.18	4.15* ± 0.69	58.68 ± 22.25
1000	10	2.74 ± 1.66	2.25 ± 2.01	4.05* ± 0.65	114.86 ± 64.22
10000	15	4.89* ± 2.47	4.97* ± 3.03	4.79* ± 0.82	242.94* ± 272.43
		Females, 6 weeks
0	15	1.28 ± 0.15	0.39 ± 0.09	2.25 ± 0.49	37.34 ± 21.62
100	10	1.25 ± 0.08	0.39 ± 0.07	2.38 ± 0.81	44.34 ± 15.88
1000	10	2.27 ± 1.67	0.84 ± 0.83	2.54 ± 0.78	82.35* ± 82.82
10000	15	2.12 ± 3.16	0.76 ± 0.75	2.38 ± 0.60	68.01 ± 30.54
		Males, 13 weeks
0	15	1.34 ± 0.14	0.60 ± 0.11	2.11 ± 0.30	55.84 ± 16.79
100	10	1.39 ± 0.13	0.70 ± 0.20	2.49* ± 0.32	61.31 ± 15.77
1000	10	1.85 ± 0.38	1.56 ± 0.70	2.37 ± 0.27	98.52 ± 44.51
10000	15	6.73* ± 3.35	8.01* ± 3.57	2.98* ± 0.47	1132.78* ± 1475.57
		Females, 13 weeks
0	15	1.23 ± 0.23	0.46 ± 0.12	1.16 ± 0.25	42.51 ± 31.39
100	10	1.19 ± 0.18	0.41 ± 0.10	1.09 ± 0.36	77.05* ± 26.76
1000	10	1.51 ± 0.54	0.48 ± 0.13	1.11 ± 0.19	47.34 ± 22.40
10000	15	1.42 ± 0.34	0.55 ± 0.22	1.15 ± 0.41	79.46* ± 36.25
		Males, after recovery
0	5	1.28 ± 0.26	0.54 ± 0.14	1.98 ± 0.10	60.01 ± 26.36
10000	5	1.18 ± 0.11	0.40 ± 0.09	2.10 ± 0.19	68.35 ± 18.86
		Females, after recovery
0	5	1.24 ± 0.09	0.44 ± 0.06	1.26 ± 0.07	54.68 ± 10.57
10000	5	1.14 ± 0.23	0.40 ± 0.05	1.36 ± 0.47	58.34 ± 28.73

* Dunnett-test based on pooled variance significant at 5% level

 

 

Selected organ weights:

Nominal concentration in feed [ppm]	Males		Females
	Liver weight [g]	Testes [g]	Liver [g]
13 weeks
0	12.82 ± 1.11	3.29 ± 0.22	7.34 ± 1.11
100	13.20 ± 1.27	3.36 ± 0.26	7.30 ± 0.76
1000	12.06 ± 1.15	3.37 ± 0.22	7.53 ± 0.80
1000	12.82 ± 1.33	3.54* ± 0.29	8.71** ± 0.61
After recovery
0	9.96 ± 0.68	3.17 ± 0.14	6.01 ± 0.89
10000	9.98 ± 1.46	3.39 ± 0.18	5.90 ± 0.65

* t-test significant at 5% level

** t-test signficiant at 1% level

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1987

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

May 12, 1981

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-(3,5-dichloro-2,4-difluorophenyl)-3-(2,6-difluorobenzoyl)urea

EC Number:

617-441-5

Cas Number:

83121-18-0

Molecular formula:

C14 H6 Cl2 F4 N2 O2

IUPAC Name:

1-(3,5-dichloro-2,4-difluorophenyl)-3-(2,6-difluorobenzoyl)urea

Test material form:

solid: crystalline

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

This test system is recognized by international regulatory agencies suitable rodent mammalian species for studies of this type.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: KFM Kleintierfarm Madoerin AG, 4414 Fuellinsdorf/Switzerland
- Age at study initiation: 4 weeks (at delivery)
- Weight at study initiation: Initial weight (pretest): males 68 - 95 g, females 67 - 96 g
- Housing: in groups of 5 in Makrolon type-4 cages with wire mesh lids
- Diet: ad libitum, pelleted standard Kliba 343 rat maintenance diet (Klingentalmuehle AG, Switzerland).
- Water: ad libitum, tap water
- Acclimation period: 10 days

DETAILS OF FOOD AND WATER QUALITY:
Certificates for the analysis of contaminants of the batches were attached to the study report.
Certificates for the analyses (chemical and bacteriological) were attached to the study report.
The food and water quality was considered to be acceptable.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: December 23, 1983 To: January 09/10, 1985 (Chronic toxicity 53 weeks)
From: December 23, 1983 To: January 23/24, 1986 (Chronic toxicity 107 weeks)

Administration / exposure

Route of administration:

oral: feed

Details on oral exposure:

DIET PREPARATION
The test substance was mixed with microgranulated feed using a Buehler type-DDMA 0.5 mixer and pelleted with a Buehler type-DFPL (Source: Buehler AG, Uzwil/Switzerland) pelleting machine. Water was added to each feed preparation at a 1:10 volume/weight ratio to ensure pelleting, after which the pellets were dried. The mixtures were prepared twice monthly and stored at room temperature in disposable paper bags.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test substance in the feed was determined during the range finding study (see cross-referenced DRF) and before commencement of the study by the sponsor.
The test substance concentrations in the diets were analysed at 12-weekly intervals by the sponsor. These results were reported separately.

Duration of treatment / exposure:

53 weeks and 107 weeks for chronic toxicity

Frequency of treatment:

continuous

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Chronic toxicity phase: males 0 mg/kg bw/day, females 0 mg/kg bw/day
Carcinogenicity phase: males 0 mg/kg bw/day, females 0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

20 ppm

Remarks:

Chronic toxicity phase: males 1.1 mg/kg bw/day, females 1.3 mg/kg bw/day
Carcinogenicity phase: males 1.0 mg/kg bw/day, females 1.2 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

100 ppm

Remarks:

Chronic toxicity phase: males 5.4 mg/kg bw/day, females 6.5 mg/kg bw/day
Carcinogenicity phase: males 4.8 mg/kg bw/day, females 5.9 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

500 ppm

Remarks:

Chronic toxicity phase: males 27.6 mg/kg bw/day, females 32.0 mg/kg bw/day
Carcinogenicity phase: males 24.8 mg/kg bw/day, females 29.9 mg/kg bw/day

No. of animals per sex per dose:

10 for 53 weeks chronic toxicity
10 for 107 weeks chronic toxicity
50 for 120 weeks carcinogenicity

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
Based on a preliminary 4-week feeding study and a previous 13-week feeding study in rats (See cross-referenced DRF studies).

- Fasting period before blood sampling for clinical biochemistry:
yes, 18 hours before sampling

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Observations for signs of toxicity were performed twice daily.
In addition to the daily observations, each rat had a weekly detailed clinical examination which included a palpation for tissue masses.

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly during weeks 1-15 and twice monthly thereafter.

FOOD CONSUMPTION:
- weekly during weeks 1-15 and twice monthly thereafter.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: after 14, 26, 53, 78, 105 and 118 weeks.
- Dose groups that were examined: 10 animals of each group and sex of the oncogenicity animals were examined.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected at 14, 26, 53, 78, 105 and 120 weeks.
- Anaesthetic used for blood collection: Yes (light ether anesthesia).
- Animals fasted: Yes (18 hours, water was provided)
- How many animals: Blood samples were collected from 20 animals of each sex and group at 14, 26, and 53 weeks, from 10 animals of each sex and group at 78 and 105 weeks, and from 20 animals of each sex and group foreseen for oncogenicity at 120 weeks.
- EDTA-K2 was used as anticoagulant during blood collection (hematology).
- Na-Citrate, 3.8% was used as anticoagulant during blood collection (coagulation, 1:10).
The following commercial reference controls were used to monitor the performance of the method:
Hematology: Control blood - level I (low range), Control blood - level II (normal range), Control blood - level III (high range) (Merz & Dade AG, Duedingen/Switzerland).
Coagulation: Ci-Trol-1 (normal range), Ci-Trol-2 (high range) (Merz & Dade AG, Duedingen/Switzerland).
The following parameters were used to determined: Erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, reticulocyte count, nucleated erythrocytes- normoblasts, total leukocyte count, differential leukocyte count, red cell morphology.
Coagulation: Thromboplastin time, partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected at 14, 26, 53, 78, 105 and 120 weeks.
- Animals fasted: Yes (18 hours, water was provided)
- How many animals: Blood samples were collected from 20 animals of each sex and group at 14, 26, and 53 weeks, from 10 animals of each sex and group at 78 and 105 weeks, and from 20 animals of each sex and group foreseen for oncogenicity at 120 weeks.
Li-heparin (140 U.S.P. Units) was used as anticoagulant during blood collection:
The following commercial reference controls were used to monitor the performance of the method:
Clinical Biochemistry: Seronorm (normal range), Pathonorm L (low range), Pathonorm H (high range)(Nyegaard & Co., Oslo/Norway).
Moni-Trol I-E (normal range), Moni-Trol II-E (abnormal range) (Merz & Dade AG, Duedingen/Switzerland).
Protein electrophoresis: Seronorm Protein (Nyegaard & Co., Oslo/Norway).
The following parameters were determined:
glucose, urea, creatinine, bilirubin total, cholesterol total, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, gamma-glutamyltransferase, ornithine carbamyltransferase, calcium, phosphorus, sodium, potassium, chloride, protein total, protein electrophoresis.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected from 20 animals of each sex and group at 14, 26, and 53 weeks, from 10 animals of each sex and group at 78 and 105 weeks, and from 20 animals of each sex and group foreseen for oncogenicity at 120 weeks.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: during the 18 hours urine collection the animals were deprived of food but allowed acces to water.
Urine samples were collected by metabolism cage. The following commercial reference controls were used to monitor the performance of the method:
Urinalysis: Chek-Stix Urinalysis Control Strips (Ames Division, Miles Laboratories, Inc., Elkhart, Indiana/USA).
The following listed parameters were determined: Volume (18 h), specific gravity, pH, protein, glucose, ketone, bilirubin, blood, urobilinogen, urine sediment.

OTHER:

HEARING TESTS:
10 animals from each sex and group with the highest identification nunbers were tested for hearing impairment using a tone generator (frequency of 10 khz, volume of 80 Db, duration of 30 ms. The test was repeated 5 times with 2-second pauses between each test). Evidence of Preyer's Reflex after 3-5 repeats constituted a positive reaction. The hearing tests were performed after 14, 26, 53, 78, 105, and 118 weeks of treatment.

PLASMA LEVEL DETERMINATIONS:
Blood samples were collected after 107 weeks of treatment (on the day of necropsy) from the surviving chronic toxicity animals for the test substance determinations in plasma.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were necropsied and descriptions of all macroscopic abnormalities were recorded. All animals which survived to the end of the observation period and all moribund animals were anesthetized by intraperitoneal injection of sodium pentobarbital and killed by exsanguination.

HISTOPATHOLOGY: Yes
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution:
Adrenal glands, aorta, brain, cecum, colon, duodenum, epididymides, esophagus, eyes with nerve and Harderian gland, heart, ileum, jejunum, kidneys, liver, lung (infused with formalin), lymph nodes (mandibular, mesenteric), mammary gland, ovaries, pancreas, pituitary gland, prostate gland, rectum, salivary gland (mandibular, sublingual), seminal vesicles, sciatic nerve, skeletal muscle, skin, spinal cord (cervical), spleen, sternum with marrow, stomach, testes, thymus, thyroid glands with parathyroid glands, tongue, trachea, urinary bladder (infused with formalin), uterus, gross lesiens, tissue masses.
All organs were trimmed, and embedded in paraffin. Sections were cut in an approximate thickness of 4 µm and stained with hematoxylin and eosin (HE).

Sections of the following tissues from all rats were examined microscopically:
Adrenal glands, aorta, bone (sternum), bone marrow (sternum), brain, epididymides, esophagus, eyes, Harderian glands, heart, kidneys, large intestine, cecum, colon, rectum, liver, lungs, lymph nodes (mandibular, mesenteric), mammary glands, ovaries, pancreas, parathyroid glands, pituitary gland, prostate, salivary glands, sciatic nerve, seminal vesicles, skeletal muscle, skin, small intestine (duodenum, jejunum, ileum), spinal cord, spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus, and all gross lesions.

OTHER:
Organ weights
The following organ weights were determined at scheduled necropsies: adrenal glands, brain, heart, kidneys, liver, ovaries, pituitary gland, spleen, testes, thyroid.

Statistics:

The following statistical methods were used to analyze body weights, food consumption, organ weights and clinical laboratory data:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
For the overall spontaneous mortality data, the Fisher's exact test for 2x2 tables was applied.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
References:
C.W. Dunnett: A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Statist. Assoc. 50, 1096-121 (1955).
R.G. Miller: Simultaneous Statistical Inference, Springer Verlag, New York (1981).
R.A. Fisher: Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The changes observed reflected the normal pattern for this strain of rat in a chronic study, and showed no relationship to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were to significant differences in mortality rate between treatment and control groups. The mortality data indicate that the duration of the study comprised the majority of the normal life-span of the animals.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

CHRONIC TOXICITY ANIMALS
Mean body weight gains were similar in all groups of females.
Mean body weight gains were slightly reduced for all groups of treated males leading to decrements in mean terminal body weights after 107 weeks of 7% for males of group 2, 6% for males of group 3 and 14% for males of group 4, relative to male controls. However, the changes noted for treated males, which are largely accounted for by unusually high body weight gains of two control males, were considered to be incidental and not significant in toxicological terms.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

CHONIC TOXICITY ANIMALS
Food consumption was similar for all groups of males as well as for all groups of females.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Incidental eye abnormalities, e.g. lens turbidity, pale and/or granulated ocular fundus were observed at an advanced age, were similarly distributed among groups, and were considered to be unrelated to treatment.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The assessment of hematological data indicated no changes of toxcological significance after 14, 26, 53, 78, 105, and 120 weeks of treatment. All statistical differences in the results of the hematological parameters were considered to be incidental and of normal biological variation.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Some treatment related changes were observed in the clinical chemistry parameters:
Slightly increased Aspartate and Alanine AminoTransferase (ASAT and ALAT) activities in males of the high dose group after 14, 26, 53 and 78 weeks and for ALAT after 120 weeks.
Slightly increased ornithine carbamyl transferase (OCT) activity for males of the high dose group after 53 and 78 weeks of treatment.

All other statistical differences in the results of the clinical biochemistry parameters were considered to be incidental and of normal biological variation.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Urinalysis data indicated no changes of toxicological significance after 14, 26, 53, 78, 105, and 120 weeks of treatment. All differences in the results of the urinalysis parameters were considered to be incidental and of normal biological variation.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The liver to body weight ratios were slighty increased (by approximately 10% on average) for males of group 4, relative to male controls, after 120 weeks of treatment. No other organ weight changes were seen during the study.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A number of gross findings were noted in rats that died during the study and in those that were sacrificed on schedule. The type and incidence of gross findings were considered to be similar in all treated groups and in the controls.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Numerous non-neoplastic lesions were diagnosed. The non-neoplastic lesions mainly affected the large parenchymatous and endocrine organs. The type, incidence, and severity of these lesions were considered to be similar in both treated rats and the control rats and within the normal spectrum for aged rats of this strain.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Interim sacrifice 53 weeks
With the exception of an incidence of 20% pituitary adenomas in females of group 1, the incidence of neoplasms in rats assigned to interim sacrifice after 53 weeks did not exceed 11.1%.

Interim sacrifice 107 weeks
Neoplastic lesions were diagnosed primarily in the pituitary gland, mammary glands, skin, and thyroid gland.
Pituitary gland:
The pituitary neoplasms were adenomas. The incidence of adenomas in males was 30% in group 1, 10% in group 2, 40% in group 3, and 10% in group 4, whereas in females the incidence was 40% in group 1, 50% in groups 2 and 3, and 70% in group 4.
Mammary gland:
The mammary gland neoplasms were fibroadenomas, fibromas, and adenocarcinomas. The incidence of mammary neoplasms in females was 33.3% in group 1, 70% in group 2, 55.5% in group 3, and 30% in group 4. In males, one fibroadenoma was diagnosed in group 4.
Skin:
The types of neoplasms diagnosed in the skin were fibroma, malignant neurinoma, sebaceous papilloma, basal cell carcinoma, and hemangioma. The incidence of skin neoplasms in males was 10% in group 1, 30% in group 2, 0% in group 3, and 30% in group 4, whereas in females the incidence was 0% in each of groups 1, 2, and 3, and 10% in group 4.
Thyroid gland:
The neoplasms of the thyroid gland were C-cell adenomas, follicular adenomas, and one C-cell carcinoma. The incidence of thyroid neoplasms in males was 22.2% in group 1, 10% in group 2, 30% in group 3, and 10% in group 4, whereas in females the incidence was 40% in group 1, 0% in group 2, 20% in group 3, and 30% in group 4.
Hemolymphoreticular system:
The neoplasms of the hemolymphoreticular system were malignant fibrous histiocytomas and one malignant lymphoma. The incidence of neoplasms of the hemolymphoreticular system was 10% in females of group 2 and 20% in males of group 3. In all other groups no hemolymphoreticular neoplasms were diagnosed.
Kidneys:
Renal neoplasms were only diagnosed in males of group 2. These renal neoplasms were 1 adenoma and one lipomatous tumor.
Liver:
Hepatic neoplasms were only diagnosed in males of group 1. These hepatic neoplasms were 1 hepatocellular adenoma and 1 hepatocellular carcinoma.
Other organs/tissues:
The incidence of rats with neoplasms in the other organs and tissues ranged from 0 to a maximum of 11.1%.

Other effects:

no effects observed

Description (incidence and severity):

Hearing tests
No hearing impairment was noted in any animal at any time interval.

Plasma level determination
The plasma level was found to be dose-related, i.e. in males, on average, levels of 0.02, 0.06 and 0.26 µg/mL were found for concentrations the test substance in the diet of 20, 100 and 500 mg/kg bw/day. The corresponding data found for females were, on average, 0.02, 0.04 and 0.15µg/mL.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

>= 4.8 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

organ weights and organ / body weight ratios

Remarks on result:

other:

Remarks:

100 ppm is equivalent to an average intake of 5.4 and 6.5 mg/kg bw/day for males and females respectively (chronic toxicity phase) and 4.8 and 5.9 mg/kg bw/day for males and females respectively (carcinogenicity phase).

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

24.8 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Any other information on results incl. tables

Test substance intake:

Nominal concentration in feed [ppm]	Average Test Substance intake [mg/kg bw/d] – 107 weeks		Average Test Substance intake [mg/kg bw/d] – 120 weeks
	Males	Females	Males	Females
0	0	0	0	0
20	1.1	1.3	1.0	1.2
100	5.4	6.5	4.8	5.9
500	27.6	32.0	24.8	29.9

 

Plasma level determination after 107 weeks of treatment:

Nominal concentration in feed [ppm]	Average Test Substance Plasma Concentration [µg/mL]
	Males	Females
20	0.02	0.02
100	0.06	0.04
500	0.27	0.15

 

Mortality rate (number of non-scheduled deaths):

Nominal concentration in feed [ppm]	Up to 107 weeks		Up to 120 weeks
	Males	Females	Males	Females
0	2	4	20	22
20	4	1	23	25
100	3	4	20	22
500	2	3	20	20

 

 

Incidence of ophthalmoscopic findings:

Treatment month	Number of animals displaying eye abnormalities
	Group 1 (0 ppm)		Group 2 (20 ppm)		Group 3 (100 ppm)		Group 4 (500 ppm)
	M	F	M	F	M	F	M	F
14	0	0	0	0	0	0	0	0
26	0	0	0	0	0	0	0	0
53	0	0	0	0	0	0	0	0
78	0	0	1	0	0	0	1	0
105	1	0	4	0	2	2	3	2
118	3	3	4	3	3	4	5	4

M – males

F – females

 

 

Selected clinical biochemistry parameters (mean ± SD):

Nominal concentration in feed [ppm]	Males			Females
	ASAT [µkat/L]	ALAT [µkat/L]	OCT [nkat/L]	ASAT [µkat/L]	ALAT [µkat/L]	OCT [nkat/L]
	14 weeks
0	1.22 ± 0.12	0.63 ± 0.11	69.83 ± 35.59	1.24 ± 0.23	0.58 ± 0.15	77.07 ± 49.95
20	1.24 ± 0.14	0.67 ± 0.16	71.68 ± 32.22	1.25 ± 0.21	0.63 ± 0.19	125.40 ± 117.54
100	1.26 ± 0.15	0.76 ± 0.18	69.51 ± 21.24	1.23 ± 0.29	0.61 ± 0.29	181.31 ± 222.78
500	1.94* ± 0.77	1.89* ± 1.33	93.85 ± 45.31	1.36 ± 0.27	0.61 ± 0.12	120.90 ± 147.03
	26 weeks
0	1.22 ± 0.12	0.61 ± 0.16	84.25 ± 40.26	1.82 ± 1.85	0.72 ± 0.81	521.04 ± 1180.08
20	1.12 ± 0.14	0.56 ± 0.16	77.96 ± 25.54	1.38 ± 0.82	0.54 ± 0.36	303.29 ± 614.55
100	1.38 ± 0.26	0.70 ± 0.28	82.71 ± 58.21	1.32 ± 0.28	0.53 ± 0.14	173.44 ± 108.71
500	1.59* ± 0.41	1.31* ± 0.73	66.57 ± 42.00	1.28 ± 0.35	0.47 ± 0.20	145.51 ± 129.84
	53 weeks
0	1.24 ± 0.22	0.79 ± 0.20	76.39 ± 56.22	1.33 ± 0.44	0.68 ± 0.36	168.82 ± 203.90
20	1.19 ± 0.21	0.82 ± 0.32	84.04 ± 57.55	1.33 ± 0.31	0.78 ± 0.46	169.60 ± 222.57
100	1.19 ± 0.15	0.74 ± 0.16	115.95 ± 115.17	1.48 ± 0.54	0.71 ± 0.21	191.36 ± 203.50
500	1.67* ± 0.37	1.77* ± 0.77	163.87* ± 117.03	1.39 ± 0.34	0.65 ± 0.16	164.34 ± 149.55
	78 weeks
0	1.56 ± 0.34	0.93 ± 0.17	49.53 ± 7.85	1.32 ± 0.16	0.72 ± 0.10	89.96 ± 37.94
20	1.52 ± 0.39	1.01 ± 0.36	87.78 ± 72.16	1.51 ± 0.49	0.87 ± 0.25	246.68 ± 281.24
100	1.47 ± 0.09	1.05 ± 0.24	96.57 ± 43.45	1.32 ± 0.17	0.75 ± 0.13	143.48 ± 93.55
500	2.22* ± 0.51	2.53* ± 1.36	159.90* ± 55.81	2.05 ± 1.61	0.75 ± 0.18	299.28 ± 411.38
	105 weeks
0	1.34 ± 0.10	0.90 ± 0.14	79.62 ± 50.51	1.33 ± 0.27	0.67 ± 0.05	88.07 ± 61.07
20	1.39 ± 0.29	0.88 ± 0.22	126.41 ± 124.27	1.70 ± 0.43	0.92* ± 0.30	289.00* ± 206.27
100	1.44 ± 0.14	0.90 ± 0.15	185.04 ± 102.33	1.40 ± 0.24	0.76 ± 0.06	198.71 ± 163.33
500	1.43 ± 0.29	0.89 ± 0.23	117.28 ± 83.37	1.50 ± 0.60	0.69 ± 0.14	104.83 ± 48.57
	120 weeks
0	1.33 ± 0.37	0.69 ± 0.11	128.23 ± 86.50	1.38 ± 0.31	0.70 ± 0.14	181.84 ± 182.87
20	1.18 ± 0.18	0.68 ± 0.13	113.94 ± 68.91	1.38 ± 0.37	0.66 ± 0.12	184.07 ± 206.61
100	1.25 ± 0.19	0.73 ± 0.13	109.74 ± 77.35	1.22 ± 0.21	0.64 ± 0.10	128.35 ± 120.70
500	1.55 ± 0.48	0.95* ± 0.37	128.33 ± 77.36	1.63 ± 0.70	0.76 ± 0.24	185.00 ± 142.48

* Dunnett-Test based on pooled variance significant at 5% level

** Dunnett-Test based on pooled variance significant at 1% level

 

 

Liver weights after 120 weeks treatment with the test substance:

Nominal concentration in feed [ppm]	Mean absolute liver weights [g] (mean ± SD)		Mean relative liver weights [%] (mean ± SD)
	Males	Females	Males	Females
0	15.87 ± 3.36	11.50 ± 1.99	2.80 ± 0.55	3.30 ± 0.40
20	16.71 ± 2.36	11.15 ± 2.17	2.88 ± 0.44	3.44 ± 0.54
100	15.13 ± 2.45	11.38 ± 2.05	2.81 ± 0.41	3.31 ± 0.38
500	17.42 ± 3.07	11.28 ± 1.70	3.12* ± 0.61	3.34 ± 0.40

* Dunnett-Test based on pooled variance significant at 5% level