2-chloro-N-[[[4-(trifluoromethoxy)phenyl]amino]carbonyl]benzamide

Carcinogenicity

Description of key information

Two pre-GLP and non-guideline chronic toxictiy studies in rat and mouse are available. No carcinogenic potential was observed.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

June 1980 - June 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of 50 male and 50 female BOR:CFW1 mice were administered Triflumuron at doses of 0, 20, 200 or 2000 ppm in the diet for periods of up to 24 months. The dietary concentrations were equivalent to a mean daily intake of 5.19, 49.0 and 523.3 mg/kg bw in the males and 6.68, 67.9 and 691.6 mg/kg bw/day in the females. Ten additional animals per sex and dose were reserved for determinations of haematological and clinical chemistry parameters after test periods of six, 12 and 18 months.

GLP compliance:

no

Remarks:

The study was performed before GLP principles were in place.

Species:

mouse

Strain:

other: BOR:CFW1 (SPF mice)

Sex:

male/female

Details on test animals or test system and environmental conditions:

The experimental animals employed were SPF mice of the BOR:CFW1 strain. The mice were approximately 5 to 6 weeks old at the beginning of the experiment. The mean initial weight of the males was 25 g, and that of the females was 23 g.

The animals were housed conventionally and singly in Type I Makrolon cages on a litter of dust-free wood shavings at a room temperature of approx. 21 - 23°C, a relative humidity of approximately 50 - 60 %, and with a 12-hour light/dark cycle (artificial illumination from 7 a.m. to 7 p.m., GET). When the cages were cleaned once a week during the experimental period, the animals were supplied with fresh pulverized feed and tap water for ad libitum consumption. The animals were identified by cage number.

Route of administration:

oral: feed

Details on exposure:

The mice received the test substance mixed with the pulverized feed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During the study diet mixtures were analyzed (9 occasions throughout the study). After extracting the diet containing the test item the concentration was measured by means of HPLC. The recovery was 81 - 120% of the nominal concentration.
Homogeneity and stability tests of the test item in diet were made. The compound was stable in the diet for 11 days. The distribution of the compound in the diet was homogeneous.

Duration of treatment / exposure:

24 months

Dose / conc.:

20 ppm

Remarks:

Low group dose, equivalent to a mean daily intake of 5.19 mg/kg bw/day in the males and 6.68 mg/kg bw/day in the females

Dose / conc.:

200 ppm

Remarks:

Mid dose group, equivalent to a mean daily intake of 49.0 mg/kg bw/day in the males and 67.9 mg/kg bw/day in the females

Dose / conc.:

2 000 ppm

Remarks:

High dose group, equivalent to a mean daily intake of 523.3 mg/kg bw/day in the males and 691.6 mg/kg bw/day in the females

No. of animals per sex per dose:

The test group for each dose level consisted of 80 male and 80 female mice. Of these, 30 mice/sex/dose were designated for laboratory tests and intermediate necropsies.

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

Observation of the Experimental Animals: The experimental animals were observed twice a day (once a day on weekends and holidays), and the changes and symptoms found were recorded.

Determination of Body Weights and Feed Consumption: The body weights of the mice were determined either weekly (Weeks 1 -14, 88 - end of study) or every 3 weeks (Weeks 15 - 87). The weekly feed consumption was determined by weighing back the unconsumed feed.


Clinical Laboratory Tests: Clinical laboratory tests were conducted on 10 males and 10 females from each test group at 6, 12, 18, and 24 months after the start of the feeding study. In addition, hematological tests were conducted at 4 months. The fluoride content in the bones and teeth of each of 5 randomly selected male and female mice from each test group was determined at 12 and 24 months. The blood for the analysis of blood glucose was obtained from the caudal vein, and from the retroorbital venous plexus of mice narcotized with ether for the remaining tests (method of NOELLER, 1955).

Haematology: Erythrocyte, leukocyte, and thrombocyte counts as well as MCV and hemoglobin concentration: measured with Coulter Counter (evaluation at 4, 6, 18, and 24 months via diluted samples), Reticulocyte count after staining with brilliant cresyl blue, Calculation of MCH, MCHC, and hematocrit, Leukocyte differential count: using smears (modified WRIGHT'S stain), Heinz bodies (only at 18 and 24 months).

Enzymes in Plasma: Alkaline phosphatase (ALP), Glutamate oxalacetate transaminase (GOT), Glutamate pyruvate transaminase (GFT).

Substrates in Plasma: Creatinine, Urea, Cholesterol, Total protein, Blood glucose, Bilirubin, Determination of fluoride concentration.

Sacrifice and pathology:

Gross pathology:

Gross Pathology of Mice that Died or Were Sacrificed when Moribund during the Experiment: Necropsy and gross examination were performed on the mice that died or were sacrificed when moribund during the experiment. The organs and tissues that still appeared amenable to evaluation were fixed in 10% formaldehyde.

Gross Pathology of Mice Sacrificed at 6, 12, and 18 Months: The mice sacrificed by diethyl ether narcosis after blood samples had been obtained were dissected and grossly examined. The weights of the following organs were determined: brain, heart, tastes, liver, lungs, spleen, kidneys, and ovaries. The following tissues from the mice were fixed in 10% formaldehyde solution: aorta, eyes, intestine (duodenum, jejunum, ileum, colon), femuren bloc with skeletal musculature and sciatic nerve, gall bladder, brain, urinary bladder, heart, testes, pituitary, salivary glands, liver (additional parts of the liver in fonnol-calcium), lungs, lymph nodes (mesenteric and nonmesenteric), stomach, spleen, adrenals, kidneys, ovaries, pancreas, prostate, seminal vesicles, sternum, (thyroid, esophagus, and trachea en bloc), and uterus, as well as all changes detected during gross examination.


Gross Pathology of Mice Sacrificed at the End of the Experiment: After 24 months of the feeding study, all surviving mice were narcotized with diethyl ether and sacrificed by exsanguination. The mice were then necropsied and grossly examined. The weights of the following organs taken from the sacrificed mice were determined: brain, heart, testes, lungs, liver, spleen, kidneys, and ovaries. The following tissues from all mice of all test groups were fixed in 10% formaldehyde solution: aorta, eyes, intestine (duodenum, jejunum, ileum, colon), femur en bloc with skeletal musculature and sciatic nerve, gall bladder, brain, urinary bladder, heart, testes, pituitary, salivary glands, liver (additional parts of the liver in formol-calcium), lungs, lymph nodes (mesenteric and nonmesenteric), stomach, spleen, adrenals, kidneys, ovaries, pancreas, prostate, seminal vesicles,
sternum, (thyroid, esophagus, and trachea en bloc), and uterus, as well as all changes detected during gross examination.

The organs and tissues of all animals of the experiment were subjected to histopathological examination.

Statistics:

The following were calculated: arithmetic means of the values of each group, standard deviation, and upper and lower confidence limits at the confidence level 1 - alpha = 95% and 1 - alpha - 99%. The data for the test populations were compared with the control population by means of the significance test (U test) of MANN, WHITNEY, and WILCOXON at the significance level alpha = 5% and alpha = 1%. The mortality rates of the test populations were compared with the control population by means of Fisher's exact test at the significance level alpha = 5% and alpha =1%,. In the tables of means in the text section of this report, significant differences from the control group are designated by one asterisk (*) for p < 0.05 and by two asterisks (**) for p < 0.01 (by + and ++ in the Appendix). The IBM-370/145 computer was used. The "Subprogram Randu" from the IBM "Scientific Subroutine Package" was used with the IBM-370 computer for generating the randomization lists.

Clinical signs:

no effects observed

Description (incidence and severity):

During the experiment, no differences from the control mice were observed in the appearance and behavior of the mice that received the test substance in the feed ration at dose levels of 20 to 2000 ppm. There were no differences in activity or in condition of coat between the mice at these dose levels.

Mortality:

no mortality observed

Description (incidence):

Treatment had no influence on the mortality of the mice.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Dose levels of up to and including 200 ppm had no influence on the body weight development of the treated mice. At the 2000 ppm dose level, there was a transitory delay in the body weight development of the male mice in comparison to control.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The male and female mice of all treated groups consumed approximately as much pulverized feed as the animals of the control group.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No pathological changes were found in the morphology of the individual cell types. There were no indications of treatment-related damage to the blood or hemopoietic tissues of males and females of the 20 ppm group. Significant differences from the control group either occurred only at isolated examination times, were not dose-related, or affected only one sex.

At the 200 ppm dose level and above, the following parameters were, for the most part, increased in comparison to control: males - reticulocyte count (during the entire experiment) , number of Heinz bodies (only at 18 and 24 months); females - reticulocyte count, number of Heinz bodies, MCH (mean corpuscular hemoglobin). In addition, 2000 ppm resulted in an increased leukocyte count and decreased erythrocyte count, as well as increased MCV, MCH, and, for the most part, increased MCHC values for males and females in comparison to control.

Other statistically significant differences from control are not regarded as toxicologically relevant since they were not dose-related or were only transitory. No toxicologically relevant shifts in the leukocyte differential count were observed. An overview of the results is included in Table 1, "Overall remarks, attachments section")

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

At 2000 ppm, the bilirubin concentration of males and females was increased in comparison to control. The other statistically significant differences from control are not regarded as toxicologically relevant, since they were not dose-related and/or occurred only in one sex.

The clinical chemistry results showed significant increases in total protein and creatinine concentrations at 12 months and increased creatinine concentrations at 18 months for treated females compared to control. These findings are not regarded as toxicologically relevant, since there was no clear dose relationship and they were not found at the end of the experiment. At the end of the experiment, females of the 200 ppm group and higher exhibited significantly lower total protein concentrations in comparison to control. However, the values were within the normal range of variation for mice of this strain, age, and sex, so that the finding is not regarded as toxicologically relevant. No pathological increases in enzyme activity were observed in the treated mice. At 24 months, the bilirubin concentration of male mice of the 200 ppm group and higher was increased in comparison to control. For the females, 2000 ppm resulted in an increased bilirubin concentration at 18 and 24 months in comparison to control.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The absolute and/or relative spleen and liver weights of male and female mice of the 2000 ppm dose group were, for the most part, higher than control at 6, 12, and 18 months. The high relative and absolute spleen weights of control females at 24 months can be attributed to the extremely high spleen weights of two female mice. For both of these mice, histopathological examination revealed neoplasms, to which the increased spleen weight can be attributed. If the spleen weights of these mice are not included in the calculation of the mean, the tendency towards increased spleen weights of females of the 2000 ppm dose group in comparison to control still remains at the end of the experiment. In comparison to control females, significant increases in mean ovary weights found in the 2000 ppm dose group at 18 months can be attributed to the extreme value for a female mouse, and in the 200 ppm dose group at 24 months to the extreme value for another female mouse. Gross examination revealed enlarged ovaries in both mice. If these extreme values, which are to be regarded as random occurrences, are not included in the calculation of the means, there are no marked differences in comparison to the respective mean of the control group females. Other significant differences in organ weights are either not dose-related or are only minimal, and are to be attributed in part to differences in body weight, and thus are not toxicologically relevant.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Mice that died during the experiment were necropsied. Gross examination revealed no changes in any of the mice that could be attributed to treatment.

The results of the gross examination of mice sacrificed at 6, 12, and 18 months gave no indications of any treatment-related organ damage at any dose level.

The results of the gross examination of all mice sacrificed at the end of the experiment revealed no indications of treatment-related organ damage at dose levels up to and including 200 ppm. For female mice of the 2000 ppm dose group, increased numbers of enlarged spleens were observed.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Increased pigment deposits in comparison to control were observed in the spleen at dose levels of 200 ppm, and in the liver and spleen at 2000 ppm. The other nonneoplastic changes were consistent with the findings to be expected for mice of this age and strain, and the conventional conditions
of their care.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

The tumors predominantly found were of the malignant lymphoma type in the reticuloendothelial system, as well as of the adenoma and carcinoma type in the lungs and liver. These types of tumors were found at approximately the same frequency in mice of all dose groups. The observed tumors were consistent with the type, number, site, and malignancy or benignity of the tumor spectrum to be expected for mice of this age and strain, and the conventional conditions of their care. The number of tumors thus shows no difference between control mice and treated mice. The number of tumor-bearing mice showed no treatment-related influence. An overview of the results is included in Table 2, "Overall remarks, attachments section").

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Determination of fluoride in bones and teeth: At 2000 ppm, statistically significant increases in fluoride concentration were found in the teeth and bones of male mice at 12 and 24 months after the start of the experiment, and, in the case of females, in the teeth at 12 months and in the bones at 24 months. The statistically significant increase in fluoride concentration found at 24 months in the case of males of the 20 ppm dose group in comparison to control is regarded as a random occurrence since it is not dose-related.

Key result

Dose descriptor:

NOAEL

Effect level:

6.68 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

haematology

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Effect level:

5.19 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

haematology

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Effect level:

20 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

200 ppm

System:

haematopoietic

Organ:

blood

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

A mouse carcinogenicity study was performed at dietary concentrations of 20, 200 and 2000 ppm. There was no evidence of carcinogenicity; the NOAEL was concluded to be 20 ppm, equivalent to 5.19 mg/kg bw/day in the males and 6.68 mg/kg bw/day in the females.

Executive summary:

Triflumuron was administered for 24 months at concentrations of 0 ppm (control), 20 ppm, 200 ppm, and 2000 ppm in the diet to groups of mice each composed of 50 males and 50 females. In addition, 10 mice/dose/sex were employed for determination of the haematological and clinical chemistry parameters at 6, 12, and 18 months. After blood samples had been obtained, the mice were sacrificed and necropsied. The mice treated at these dose levels did not differ from the control mice in appearance or behaviour, feed consumption, or mortality. There was no influence on the body weight development of mice treated at dose levels of up to and including 200 ppm. The 2000 ppm dose resulted in a transitory delay in body weight development, predominantly in male mice. Haematological, clinical chemistry, and histopathological tests and examinations gave indications of blood damage in the form of haemolytic anaemia at dose levels of 200 ppm and higher. Clinical chemistry, gross pathology, and histopathology findings provided no evidence of liver damage in dose groups up to and including 2000 ppm. Clinical chemistry, gross pathology, gravimetric organ findings, and histopathological results provided no evidence of kidney damage in the dose groups up to 2000 ppm. The 2000 ppm dose level resulted in slightly increased fluoride concentrations in the bones and teeth of treated mice. In regard to the other examined organs, necropsy and the findings of the gross pathological and histopathological examinations of mice that died during the experiment, of mice that were sacrificed when moribund, of mice employed for the intermediate necropsies at 6, 12, and 18 months, and of those sacrificed at the end of the experiment revealed no evidence of a specific organ-damaging effect at dose levels of up to and including 2000 ppm. The observed neoplastic changes were consistent with the tumour spectrum to be expected for mice of the age and strain employed and the conventional conditions of their care. No oncogenic effect was observed in any of the dose groups. Thus, under the experimental conditions described, the no observable effect level for the 24-month administration of triflumuron in the diet of male and female mice was 20 ppm.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

April 1980 - April 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Triflumuron was administered to groups of 50 male and female BOR:WISW rats at concentrations of 0, 20, 200 or 2000 ppm in the diet for periods of up to two years. The dietary concentrations were equivalent to a mean daily intake of 0.82, 8.45 and 86.1 mg/kg bw in the males and 1.11, 11.2 and 110 mg/kg bw/day in the females. Ten additional animals per sex and dose group were reserved for an intermediate necropsy after one year. The identity of the test compound and its homogeneity and stability in the feed were controlled over the period involved. Analyses for the test compound content in the feed confirmed the concentrations. Full histopathology was performed on the rats sacrificed after 12 months and after 24 months.

GLP compliance:

no

Remarks:

The study was performed before GLP principles were in place.

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

The experimental animals employed were SPF rats of the BOR:WISW strain (SPF Cpb) (bred by Winkelmann, Borchen, Federal Republic of Germany). The rats were approx. 30 to 35 days old at the beginning of the experiment. The mean initial weight of the males and females was 80 g. The animals were housed conventionally and singly in Type II Makrolon cages on a litter of dust-free wood shavings at an average room temperature of 22°C, an average relative humidity of 55%, with temporary increases up to 90% (particularly associated with the cleaning of the animal quarters), and with a 12-hour light/dark cycle (artificial illumination from 7 a.m. to 7 p.m., GET). When the cages were cleaned once a week during the experimental period, the animals were supplied with fresh pulverized feed (manufactured by Altromin GmbH, Lage, Federal Republic of Germany) and tap water for ad libitum consumption. The animals were identified by cage number.

Route of administration:

oral: feed

Details on exposure:

The rats received the test substance mixed with the pulverized feed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Feed samples were analysed using a validated method. The concentration of the test item in the animal feed was determined at 8 different times during the study. The mean recovery was 89% - 92% of the nominal concentration.
The distribution of the test item was homogeneous, since the relative standard deviation of the individual values was less than 10X (95% - 103%) (data from a study that was run in parallel to this study).
The test compound was stable in the feed mixture over a period of 7 days in consideration of the tolerance range of ±20% relative to the nominal concentration (actual: 87% - 93%).

Duration of treatment / exposure:

2 year study

Frequency of treatment:

Continuous exposure via feed.

Dose / conc.:

20 ppm

Remarks:

Low dose group, equivalent to a mean daily intake of 5.19 mg/kg bw/day in the males and 6.68 mg/kg bw/day in the females

Dose / conc.:

200 ppm

Remarks:

Mid dose group, equivalent to a mean daily intake of 8.45 mg/kg bw in the males and 11.2 mg/kg bw/day in the females

Dose / conc.:

2 000 ppm

Remarks:

High dose group, equivalent to a mean daily intake of 86.1 mg/kg bw/day in the males and 101 mg/kg bw/day in the females

No. of animals per sex per dose:

The test group for each dose level consisted of 50 male and 50 female rats. An additional 10 rats/ dose/sex were employed for an intermediate necropsy after one year.

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

Observation of the Experimental Animals: The experimental animals were observed twice a day (once a day on weekends and holidays), and the changes and symptoms found were recorded.

Determination of Body Weights and Feed Consumption: The body weights of the rats were determined at the start of the study, weekly through Week 27, and every 14 days thereafter. The weekly feed consumption was determined by weighing back the unconsumed feed.

Clinical laboratory tests: Conducted on 10 males and 10 females from each test group at 6, 12, 18, and 24 months after the start of the feeding study. The blood for the analysis of blood glucose concentration was obtained from one of the venae caudales of non-narcotized rats, by cardiac puncture for the determination of the thromboplastin time at the end of the experiment, and from the retroorbital venous plexus of rats narcotized with ether for the remaining tests . The urine samples were collected over a period of approx. 16 hours, during which tap water was available to the rats ad libitum. The rats were fasted during this period.

Urinalyses:

Semiquantitative: Glucose, blood, protein, and pH using Combi-Uristix and Bili-Labstix test reagents, Ketone bodies using Ketostix and Bili-Labstix test reagents, Bilirubin using Ictostix and Bili-Labstix test reagents, Urobilinogen using Urobilistix, Microscopic examination of the sediment after centrifuging the urine samples was carried out.

Quantitative: Analysis carried out for the protein.

Determination of Fluoride Accumulation: A determination of fluoride in the teeth and bones was conducted on 5 rats/sex of the 2000 ppm group and of the control at 12 months and on 5 rats/sex of all groups at the end of the study.

Sacrifice and pathology:

Gross Pathology:

Gross Pathology of Rats that Died or Were Sacrificed when Moribund during the Experiment:
Necropsy and gross examination were performed on the rats that died or were sacrificed when moribund during the experiment. The organs and tissues that still appeared amenable to evaluation were fixed in 10% formaldehyde.

Gross Pathology of Rats Sacrificed at 12 Months and at the End of the Experiment:

After 12 months of the feeding study, 10 rats/sex/dose (at the end of the experiment, all surviving rats) were narcotized with diethyl ether and sacrificed by exsanguination. The animals were then dissected and grossly examined. The weights of the following organs taken from the sacrificed rats were determined: brain, heart, testes, lungs, liver, spleen, kidneys, adrenals, ovaries, and thyroid.

The following tissues from all rats of all test groups were fixed in 10% formaldehyde solution: aorta, eyes, intestine (duodenum, ileum, jejunum, colon), femur en bloc with skeletal musculature and sciatic nerve, brain, urinary bladder, heart, testes, pituitary, salivary glands, liver, lungs, lymph nodes, stomach, spleen, adrenals, kidneys, esophagus, ovaries, pancreas, prostate, seminal vesicles, thyroid, sternum, thymus (if present), trachea, and uterus, as well as all changes detected during gross examination.

Other examinations:

Histopathological Examinations:

Scope of the Histopathological Examinations To the extent they were amenable to evaluation, the organs and tissues of all animals of all groups were subjected to histopathological examination.

Statistics:

The following were calculated: arithmetic means of the values of each group, standard deviation s, and upper and lower confidence limits at the confidence level 1 - alpha = 95% and 1 - alpha = 99%. The data for the test populations were compared with the control population by means of the significance test (U test) of MANN, WHITNEY, and WILCOXON at the significance level alpha = 5% and alpha = 1%. The mortality and tumor rates of the test populations were compared with the control population by means of Fisher's exact test at the significance level alpha = 5% and alpha =1%.

Clinical signs:

no effects observed

Description (incidence and severity):

During the experiment, no differences from the control rats were found in the appearance and behavior of the rats that received the test item in the feed ration at dose levels up to and including 2000 ppm. There were no differences in activity or in condition of coat between the rats at these dose levels.

Mortality:

no mortality observed

Description (incidence):

Treatment had no influence on mortality.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no dose-related retardation in body weight development in comparison to control.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The male and female rats of all treated groups consumed approximately as much pulverized feed as the animals of the control group.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Six months after the start of the study, there were no toxicologically significant, dose-related differences between rats of the 20 ppm group and control either with respect to erythrocyte, leukocyte, and thrombocyte counts or hemoglobin concentration and hematocrit. In addition, no differences attributable to treatment were found between the treated groups and control with respect to mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC).

In the 2000 ppm dose group, the erythrocyte count, hemoglobin concentration and MCHC were lower, and the reticulocyte count and MCV were higher. A lower hematocrit and higher MCH (females) and a higher leukocyte count (males) were each detected in rats of one sex only. Male rats of the 200 ppm group also had a lower hemoglobin concentration and MCHC and a higher reticulocyte count. All differences from control described above were statistically significant. The data on the leukocyte differential count indicate that treatment had no significant influence on the percentage of individual cell types. Nor were any pathological changes found in the morphology of the individual cell types in the groups up to and including 200 ppm. In the 2000 ppm group, there was an increased incidence of anulocytes; several rats also had increased polychromatophilia.

For both sexes of the 2000 ppm group, the leukocyte count and reticulocyte count were higher and the erythrocyte count was lower at the test times indicated. The thrombocyte count was
higher at 12 months in the case of females and at the end of the study in the case of males. In addition, the hemoglobin concentration, hematocrit, and MCHC were lower and the MCV and MCH were higher. In the 200 ppm group as well, deviations from control were found at all test times in some of these parameters (leukocyte, erythrocyte, and reticulocyte counts, hemoglobin concentration, hematocrit, MCV, and MCHC), predominantly in the case of females. The percentage of monocytes was significantly lower at 12 months (females), and the percentage of lymphocytes was higher at 18 months (males). Increased polychromatophilia and anisocytosis (all test times), increased anulocytes (12 and 18 months), and Heinz bodies and/or Howell-Jolly bodies (18 months) were detected for the 2000 ppm group. With the exception of the inclusion bodies, the 200 ppm group also exhibited similar findings at 18 months. The results are attached below in tabular form.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The data cited indicate no relevant and/or dose-related differences between the 20 ppm dose group and control. On the other hand, females of the 2000 ppm group had higher GOT activities, and males of the 2000 and 200 ppm groups had lower GOT and GPT activities as well as lower creatinine concentrations. These statistically significant differences from control are not, however, regarded as toxicologically relevant; each involved only one sex and, with the exception of the GOT activities of the males, were within the range of normal variation. In addition, the GOT and GPT activities of the male control rats were relatively high and were predominantly higher than the normal historical data. The clinical chemistry data cited show no treatment-related differences between the treated groups and control. The statistically significant differences from control of several parameters
(bilirubin, urea, blood glucose, protein in the plasma, creatinine, cholesterol, GOT, GPT), which involved only one sex or the other in each case, and for which the concentrations or activities were within the range of normal variation, are also to be regarded as random occurrences.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The quantitative determination of protein in the urine indicated no treatment-related differences between control and the treated groups. The results of the urinalyses performed on 10 rats/sex/dose at 6, 12, 18, and 24 months revealed no differences between the rats treated at any dose level and the control rats. No glucose, ketone bodies, or bilirubin were found in the urine of any of the rats tested. Positive results for protein and blood in the urine were present at approximately the same frequency in all groups. There was no relevant difference from control in the urobilinogen content and pH of the treated animals. The examination of the sediment did not indicate any treatment-related influence.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Both males and females of all dose groups had higher spleen weights at 12 months. In addition, males of the 2000 ppm dose group had higher heart weights than control. At the end of the experiment, the organ weights in the groups up to and including 200 ppm showed no relevant and/or dose-related differences from control. Both male and female rats of the 2000 ppm dose group had higher spleen weights, and a lower heart weight was also recorded for the females. Higher spleen weights were calculated for both sexes of this group (and also for females of the 200 ppm dose group), and higher lung, kidney, and ovary weights were found for females only. All differences from control cited were statistically significant. The remaining differences, also statistically significant but not dose-related, will be found in the tables.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross Pathology of Rats that Died or Were Sacrificed when Moribund during the Experiment: Gross examination revealed no changes in any of the rats that could be attributed to treatment.

Gross Pathology of Rats Sacrificed at 12 Months and at the End of the Experiment: The results of the gross examination of rats sacrificed at 12 months and at the end of the experiment gave no indications of any treatment-related damage at dose levels up to and including 200 ppm. For rats of the 2000 ppm dose group, enlarged spleens were observed at both examination times.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The examined organs of the rats of all groups showed normal senile changes which were consistent with the age of the rats. Increased deposits of pigment (hemosiderin) were detected in the spleen and liver of rats of the 2000 ppm dose group. Hemopoiesis in the spleen was increased.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Mainly tumors of the pituitary and thyroid of the adenoma type were found in both the control and the dose groups, and in the male rats Leydig's cell tumors in the testes, and in the females stromal polyps in the uterus. The number of pheochromocytomas (20 and 2000 ppm group) in the male rats and the total malignant tumors (200 and 2000 ppm group) in the females, and in the number of tumor-bearing females with benign and malignant tumors (2000 ppm) and, without dose correlation, the number of tumor-bearing females with malignant tumors (200 ppm group) differed statistically significantly (Fisher's exact test) from the controls. All the incidences, including the higher ones listed, were however within the range of variation for the rat strain used. The results are attached below in tabular form.

Relevance of carcinogenic effects / potential:

No carcinogenic potential.

Key result

Dose descriptor:

NOAEL

Effect level:

6.68 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

haematology

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Effect level:

5.19 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

haematology

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Effect level:

20 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

200 ppm

System:

cardiovascular

Organ:

blood

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

A rat carcinogenicity study was performed at dietary concentrations of 20, 200 and 2000 ppm. There was no evidence of carcinogenicity; the NOAEL was considered to be 20 ppm, equivalent to a mean daily intake of 5.19 mg/kg bw/day in the males and 6.68 mg/kg bw/day in the females.

Executive summary:

Triflumuron was administered for up to 2 years at concentrations of 0 ppm (control), 20 ppm, 200 ppm, and 2000 ppm in the diet to groups of rats each composed of 50 males and 50 females. In addition, 10 rats/dose/sex were employed for an intermediate necropsy after one year. During the entire study, the rats treated at these dose levels did not differ from the control rats in  appearance or behaviour. Nor was there any treatment-related influence on feed consumption, body weight development, or mortality. Haematological tests provided evidence of an influence on the blood at dose levels of 200 ppm and 2000 ppm. In correlation with this, but only at the 2000 ppm dose level, were higher spleen weights, increased hemosiderin deposits in the spleen and liver, and increased hemopoiesis in the spleen. Clinical chemistry, gross pathological, and histopathological examinations provided no evidence of a treatment-related influence on the remaining organs and tissues. At the 2000 ppm dose level, increased fluoride concentrations were found in the teeth and bones. The first indications of accumulation were also obtained at the 200 ppm dose level. The type, site, and frequency of the tumours found gave no indication of an oncogenic effect. In summary, the administration of 200 ppm and 2000 ppm Triflumuron did have an influence on the blood picture and also resulted in evidence of increased fluoride accumulation. The no observable effect level was 20 ppm.  

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

0.82 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Two reliable (Klimisch 2) chronic toxicity studies are available in the rat and mouse.

System:

haematopoietic

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

In two chronic toxicity studies with rats and mice indications of haemolytic anaemia were found in haematology, necropsy and histopathology. As a conservative approach for the 2-year rat study, the equivocal haematological changes observed at the mid dose and accompanied by an increased spleen weight and pigment deposits were considered as adverse, resulting in an agreed NOAEL of 0.82 mg/kg bw/day. Similarly in mice, the agreed NOAEL was 5.19 mg/kg bw/day based on haematological effects (in both sexes) and increased bilirubin (males). With regard to the carcinogenic properties of the compound, there was no indication of an oncogenic potential. Therefore, triflumuron does not meet the criteria for classification for carcinogenicity according to Regulation (EC) No. 1272/2008.

Justification for classification or non-classification

Based on the current data-set, triflumuron is not classificed for carcinogenicity according to Regulation (EC) No. 1272/2008.