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

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 August 1979 - 28 November 1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1981

Report date:

1981

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

A 90-day feeding study was performed in male and female rats with triflumuron at 0, 50, 500 or 5000 ppm in the feed. Parameters included clinical signs, body weight, food and test compound intake, hematology, clinical chemistry, urinalysis, gross necropsy and histopathology.

GLP compliance:

no

Limit test:

no

Test material

Test material information

1

Reference substance name:

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

EC Number:

264-980-3

EC Name:

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

Cas Number:

64628-44-0

Molecular formula:

C15H10ClF3N2O3

IUPAC Name:

3-(2-chlorobenzoyl)-1-[4-(trifluoromethoxy)phenyl]urea

Test animals

Species:

rat

Strain:

Wistar

Remarks:

W 74

Details on species / strain selection:

Standard laboratory animal species/strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

At the start of the study the rats were 4 to 50 days old. The male animals had a mean starting weight of 95 g, and the females 90 g. They were kept conventionally and singly in Makrolon cages type II on dust-free wood granulate at a room temperature of 23 ± 2 °C, humidity 60 ± 5% and with a 12-hour light/dark rhythm (artificial lighting from 7 am to 7 pm). During the study period all the animals were given fresh Altromin R powdered feed once a week, and tap water ad libitum.

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The test substance was mixed in the rats' powdered feed.

Analytical verification of doses or concentrations:

no

Frequency of treatment:

Continuous exposure via feed.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 ppm

Remarks:

Low dose group, equivalent to 3.6 mg/kg bw/day for males and 4.5 mg/kg bw/day for females

Doses / concentrations 2

Dose / conc.:

500 ppm

Remarks:

Mid dose group, equivalent to 35.5 mg/kg bw/day for males and 47.0 mg/kg bw/day for females

Doses / concentrations 3

Dose / conc.:

5 000 ppm

Remarks:

High dose group, equivalent to 359.2 mg/kg bw/day for males and 448.7 mg/kg bw/day for females

No. of animals per sex per dose:

20

Control animals:

yes, concurrent no treatment

Examinations

Observations and examinations performed and frequency:

General observations: The animals were inspected daily.

Determination of the body weights and food intake: The animals were weighed weekly. The weekly food intake was determined.

Clinical lab examinations: Clinical lab examinations were conducted one and three months after start of feeding on five male and five female rats in each dose group. The blood for the blood sugar test was taken from one of the venae caudales, to obtain the thromboplastin time at end of study by heart puncture, and for the other determinations from the retro-orbital venus plexus of ether-narcotized animals.

Haematological examinations: Erythrocyte, leucocyte, and thrombocyte count by Coulter Counter reticulocyte count after staining with brilliant cresyl blue haemoglobin level and MCV with Coulter Counter differential blood count by smears (stained by Wright's method) MCH, MCHC and haematocrit calculation thromboplastin time at the end of the study by QUICK'S method (1951).

Clinical-chemical examinations:
Enzymes: alkaline phosphatase (ALP), glutamate oxalacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), glutamate dehydrogenase (GLDH, only at end of study).

Substrates: Creatinine, Urea, blood sugar, cholesterol according, bilirubin, and total protein in the plasma.

Urinalyses: Urine was collected over a period of about 16 hours, at the beginning of which each animal received approximately 5 ml tap water by stomach tube.

Semi-quantitative : Glucose, blood, protein and pH figure with Combi-Uristix, ketone bodies with Ketostix, bilirubin with Ictostix, urobilinogen with Urobilistix, microscopic deposit test after centrifuging urine.

Quantitative: Protein.

Sacrifice and pathology:

Gross pathology:

a) Rats dying during the study: These rats were dissected and grossly appraised, and the organs and tissues which were still capable of evaluation were fixed in 10 % formaldehyde solution.

b) Rats sacrificed at end of study: At the end of the three-month feeding period all the surviving animals were narcotized with ether and sacrificed by exsanguination. The animals were then dissected and grossly appraised.

The following organs were weighed at sacrifice: thyroid, heart, lung, liver, spleen, kidneys, adrenals, testicles and ovaries, and thymus. The following organ material from five male and five female rats in each dose group of the animals sacrificed at the end of the study was fixed in Bouin's fluid: aorta, eyes, intestine, femur, brain, urinary bladder, heart, testicles, pituitary, oral salivary glands, liver, lung, lymph nodes, stomach, spleen, epididymis, adrenals, nervus-ischiadicus, kidneys, oesophagus, ovaries, pancreas, prostate, seminal vesicle, thyroid, skeletal musculature, sternum, thymus, trachea and uterus, also all alterations grossly apparent. For the fat demonstration one liver lobe from each rat was fixed in formol-Ca. The organs weighed, and also brain, pituitary, uterus and in addition all the altered organs and tissue from the remaining animals were fixed in 10 % formaldehyde solution.

Statistics:

The following were calculated: arithmetic group means, standard deviations, upper and lower confidence limits at the confidence level of 1 - alpha = 95 % and 1 - alpha = 99 %. The values for the test groups with the doses investigated were compared with the control group with MANN, WHITNEY and WILCOXON's significance test (U test) at the significance level alpha = 5 % and alpha = 1 %. An IBM-370/145 computer was used. The random list was produced with the aid of the Subprogram Randu from the IBM Scientific Subroutine Package on the IBM-370 computer.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the study period the rats receiving the test item at 0 to 500 ppm in their feed did not differ in appearance and behaviour from the control animals. For the rats in these dose groups there were no differences in liveliness and coat condition, or in their eating and drinking requirements.
Males in the 5000 ppm group were subject to severe spasms lasting about 15-30 minutes during the entire study period, and the spasms sometimes occurred several times daily. After the spasms had subsided, a number of these rats had physical head injuries and bled at the nose.

Mortality:

mortality observed, treatment-related

Description (incidence):

During the entire study period, seven (four male and three female) rats died in the 5000 ppm group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The male rats in the 50 to 500 ppm study groups gained weight at about the same rate as the control animals (Table 2, "Overall remarks, attachments section"). In the 5000 ppm group the body weight trend was retarded by about 10-15 % during the entire study period. Female animals in the 500 and 5000 ppm groups had slightly, but significantly lower body weights (about 7%) up to the sixth study week compared with the control. In the second half of the study, no further significant body weight differences compared with the controls were observed in these dose groups. At 50 ppm female rats showed a slight (up to 5 %) temporarily significantly retarded body weight development during the entire study period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The male and female rats administered the test item up to 500 ppm in their feed consumed about as much powdered feed as the control animals. Male animals in the 5000 ppm group, however, consumed about 14% less food. (Table 1, "Overall remarks, attachments section")

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The treated rats in the 50 ppm dose group did not differ significantly one month after start of study from the controls, either in erythrocyte, reticulocyte, leucocyte and thrombocyte count, or in haemoglobin and haematocrit levels. (Table 3a, "Overall remarks, attachments section") There were also no differences compared to the controls in this study group in MCH value and mean cell volume (MCV). With female animals in this group, slight (about 3 %) but significantly lower MCHC values were obtained. For all the animals in the 5000 ppm group, compared to the control higher reticulocyte and leucocyte counts and lower haemoglobin levels were noted. For the differential blood count no dose-related differences in the proportions of the various cell forms were noted between the treated animals in the study groups up to 5000 ppm and the controls. (Table 3b, "Overall remarks, attachments section") The structure of the various cell types also did not produce any pathological findings.

The treated rats in the 50 ppm group did not differ significantly from the controls in any of the parameters investigated at the end of the study. (Table 4a, "Overall remarks, attachments section")
At 5000 ppm, lower erythrocyte counts and lower haemoglobin and haematocrit levels, also higher reticulocyte counts were noted for male and female animals. In addition, female rats in this group had higher MCH and MCV values. The higher leucocyte counts with the animals of both sexes were however only statistically significant for the females. At 500 ppm lower haemoglobin and haematocrit values were obtained for all the animals. In addition, the erythrocyte count was lower and the MCV value was higher for female animals than for the controls.
On the basis of the differential blood count, no treatment induced alterations in the leucocytary blood count were noted in the dose groups up to 500 ppm. (Table 4b, "Overall remarks, attachments section") Compared with the control group however, the count for polymorphonuclear granulocytes in the 5000 ppm dose group was higher, and that for the lymphocytes lower.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The findings of the urinalyses after one month of the study and at end of study for five male and five female rats in each case, did not produce any differences between control animals and treated rats up to the 5000 ppm study group. (Table 49 to 52, "Overall remarks, attachments section") No glucose, ketone bodies or bilirubin was found in the urine of any of the rats examined.
Protein and positive blood urine findings were present in all groups with about the same frequency. The urobilinogen level and pH of the urine of the treated animals did not differ significantly from that of the control animals.
The sediment examination did not reveal any effect related to the treatment. The mean urea and creatinine concentrations and the quantitatively determined protein levels in the urines, established for five male and five female rats in each case one and three months after start of study. (Table 7, "Overall remarks, attachments section")
The urine protein levels were also not pathologically increased in any dose group. After one study month lower creatinine concentrations compared with the controls were only recorded for female rats in the 500 ppm and 5000 ppm groups.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Compared with the control, animals in the 5000 ppm group had lower thymus and higher spleen and adrenal weights. Female animals in this group, in addition, had higher liver weights. After 500 ppm rats of both sexes likewise had higher spleen weights. Moreover, for male animals in this group the adrenals were heavier, and for female animals the thymus lighter than in the case of the controls.

Compared with the controls, for both sexes in all the dose groups higher lung, liver and adrenal weights were observed. These differences are partially significant. The relative spleen weights for male and female rats were also higher, however only in the groups with 500 and 5000 ppm. In addition, after 5000 ppm higher heart and kidney weights and lower thymus weights were observed in male animals.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Rats that died during the study: All the rats in the 5000 ppm group that died during the study were dissected. The enlarged or swollen spleens of two male rats, could be considered results of the treatment.

Rats sacrificed at end of study: Gross pathology did not reveal any specific alterations in the study groups up to 500 ppm in any of the rats sacrificed at the end of the study. Animals in 5000 ppm group had enlarged or swollen spleens.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathological findings for the rats that died during the study: The fixed organ material from 4 males and 3 females in the highest dose group (5000 ppm) was evaluated histopathologically. The findings were congestion of the spleen, heightened haematopoiesis in the bone marrow, and in the lung up to moderate cellular infiltrates. Cerebral extravasations were noted for one female.

Histopathological findings for the rats sacrificed at end of study: The rats' spleens in the highest dose group (5000 ppm) had no follicles. For animals in the two top dose groups (5000 and 500 ppm), in addition, the ionised iron content (Turnbull blue method) was increased, the splenic pulp sinusoids were hyperaemic, and extramedullary haematopoiesis was heightened. The bone marrow of the animals in these two groups likewise exhibited heightened blood cell formation ("full marrow"). In the liver of the rats in the highest dose group (5000 ppm) Kupffer's cells with a heightened iron content were found. The other findings given were present to about the same extent in both the control and the treatment group animals. An overview of the results is included in Tables 70 to 77, "Overall remarks, attachments section")

Details on results:

In the groups up to 5000 ppm no dose-related differences compared to the control were noted for the parameters investigated.

The activities of alkaline phosphatase (ALP), the transaminases GOT and GPT, and the glutamate dehydrogenase (GLDH, only determined at end of study) do not indicate significant differences between the treated rats in the groups up to 500 ppm and the control animals. The bilirubin and total protein concentration in the plasma also do not differ significantly for these study groups. All the figures are within the normal range. After 5000 ppm, for male rats significantly lower total protein concentrations were recorded. With female rats the glutamate dehydrogenase concentrations were significantly lower and the bilirubin values significantly higher than with the control.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Effect level:

3.6 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

3

Key result

Dose descriptor:

NOAEL

Effect level:

4.5 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 ppm

System:

haematopoietic

Organ:

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Male and female rats received the test item in concentrations of 0, 50, 500 and 5000 ppm for three months in their food. During the entire study period, the animals fed in this way in the dose groups up to 500 ppm showed no changes in appearance and behaviour. Mortality in these groups was also not affected by the treatment. The spasms, sometimes occurring several times daily, with male rats in the 5000 ppm group are considered a consequence of the treatment; the mortality
rate was also increased in this group as a result of the treatment.
The intake of food was not affected for male and female rats in the dose groups up to 500 ppm. After 5000 ppm male rats, however, consumed about 14 % less food than the control animals. The partly significant body weight differences observed for female animals after administration of 500 and 50 ppm compared with the control are, in spite of the ten-times higher dose, almost identical or minimal, and are regarded as chance findings. In the 5000 ppm dose, the body weighttrend was significantly retarded for male rats during the entire study period, and for females during the first half of the study.

After one study month significantly lower MCHC values were noted for female animals in the 50 ppm group. These differences compared with the control were however minimal (3 %), occurred only in one sex, in addition all were entirely within the biologically determined spread, and are considered toxicologically insignificant.

In the 500 and 5000 ppm dose groups at end of study, and the 5000 ppm group after one study month, lower erythrocyte counts and lower haemoglobin and haematocrit figures, and higher MGV and MCH figures were obtained. Moreover, at both points in time the reticulocyte counts in the 5000 ppm group were higher. These values, which deviate significantly from those of the controls, indicate haemolytic anaemia. The heightened haematopoiesis (spleen, bone marrow) and the increased presence of ionised iron (spleen, liver) detected by the histopathological examinations are interpreted as a symptom of compensation and as a consequence of the anaemia. The hyperaemia of the spleen sinusoids must be a consequence of the increased presence of ageing or immature erythrocytes in circulating blood possible with anaemia, as in such a case the flow of blood through the spleen is retarded.

As lengthy spleen congestion may result in atrophy of the folicles (JUBB and KENNEDY 1968), it is therefore likely that the spleen follicle atrophy is also a consequence of anaemia. In addition, the higher leucocyte counts and the higher number of polymorphs and lower number of lymphocytes, observed at doses of 5000 and 500 ppm, may also be due to the anaemia.
At the end of the study, compared with the control, the protein concentrations were lower in the dose group of 5000 ppm for male rats, and for the females the glutamate dehydrogenase activities were lower and the bilirubin concentrations higher.
These differences occurred, however, in each case only with one sex, and lay within the biologically determined spread (2s range: total protein 55-5 - 69-9 g/l, glutamate dehydrogenase 0-18.4 U/l, bilirubin 1.7 - 4.5 μmol/l).
The higher relative liver weights calculated for female rats after 50 and 500 ppm are considered a consequence of the animals' lower weights. The higher liver weights observed (female) and calculated (male and female) for animals in the
5000 ppm group may be considered the result of a minor secondary effect on the liver, a consequence of the anaemia detected by haematology. Clinical chemistry and histopathology produced, however, no indications of liver damage.
The heightened presence of ionised iron in the liver of animals in the 5000 ppm dose established by histopathology is interpreted as a consequence of the primary anaemia. There is therefore no indication that the test substance
has a hepatotoxic effect.
In the 500 and 5000 ppm dose group lower creatinine concentrations compared with the control were obtained for female rats after one study month. These differences occurred, however, only for this one examination and for one sex, and
the results were all within the range of physiological values for rats of this age (2s range 28-92 μmol/l). Gross pathology, organ weights and histopathology produced no indications of damage to the kidneys induced by the treatment. The higher relative kidney weights calculated for male animals in the 5000 ppm dose group are also not considered an indication of treatment-induced damage, but rather as a result of the significantly lower body weights in this group. The test item therefore caused no kidney damage. Blood sugar and cholesterol concentrations, also those significantly higher compared with the controls (female rats in the 5000 ppm group at end of study), all lay within the physiological range (2s range cholesterol 0.88 - 2.08 μmol/l) in all the dose groups for all the examinations
undertaken.
The enlarged or swollen spleens of the rats dying during the study, also the higher spleen weights at end of study are regarded with the findings of histopathology already discussed as a consequence of anaemia. Gross pathology and histopathology produced no indications of treatment-induced damage to the other organs. The significant organ weight differences compared with the controls obtained for adrenals and thymus and the calculated weights for thymus, heart,
lung, adrenals, testicles and ovaries must accordingly have no toxicological significance. Moreover, the differences between calculated organ weights and the controls were a consequence of the low animal weights (male rats) and/or only
occurred for one sex or unrelated to dose.
Under the conditions described, the test item was therefore tolerated without damage by rats at a dose of 50 ppm.

Applicant's summary and conclusion

Conclusions:

Based on the results of a 90-day feeding study in rats, the NOAEL is concluded to be 50 ppm, corresponding to 3.6 and 4.5 mg/kg bw/day, respectively.

Executive summary:

Groups of 20 male and 20 female rats received triflumuron for three months at dietary concentrations of 0 (control), 50, 500 and 5000 ppm. Parameters included clinical signs, body weight, food and test compound intake,hematology, clinical chemistry, urinalysis, gross necropsy and histopathology. Appearance, behaviour and mortality were not affected in male and female rats receiving doses up to 500 ppm.  At 5000 ppm, male rats showed severe treatment-induced spasms lasting about 15-30 minutes, which sometimes occurred several times daily. The mortality rate was increased in this dose group.  Food intake of male and female rats up to 500 ppm was not affected. Male rats in the 5000 ppm group consumed about ~14% less food.  Growth was not affected up to 500 ppm. At 5000 ppm, weight gains were retarded for male rats throughout the entire study, and for females in the first half of the study.  Haematology did not reveal any effects of treatment at up to 50 ppm.  At 500 and 5000 ppm, signs of haemolytic anaemia were observed.  Clinical chemistry, gross and microscopic pathology detected no liver damage in male and female rats dosed up to 500 ppm. Higher liver weights noted for animals at 5000 ppm group are interpreted as a minor secondary effect. Clinical chemistry and histopathology did not reveal indications of liver damage; the increased presence of ionised iron in the livers of animals in this group is interpreted as a consequence of anaemia. Urinalysis, clinical chemistry analysis, necropsy and histopathology showed no indication of treatment-induced kidney damage up to 5000 ppm.  Blood sugar and cholesterol concentrations were within the normal ranges for rats up to 5000 ppm.
Gross and histopathological examinations of the remaining organs provided no indications of treatment-induced organ changes up to 5000 ppm. Doses of 50 ppm were tolerated, under the conditions described without any effect. The NOAEL for this study is therefore 50 ppm (3.6 and 4.5 mg/kg bw/d in males and females, respectively)