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

Endpoint summary

Description of key information

The key studies for repeated dose toxicity endpoints are as follows:
90-day oral (dietary) rat study (no guideline, pre-GLP, RL2), NOAEL = 3.6 mg/kg bw/day (M- 087617-01-1, Krotlinger, 1981)
12-month oral (dietary) dog study (EPA 83-1, pre-GLP, RL2), NOAEL = 1.42 mg/kg bw/day (M- 087430-02-1 and M-087261-02-1, Hoffmann, 1984/89)
21-day dermal rabbit study (OECD 410, GLP, RL1), NOAEL = 100 mg/kg bw/day (M- 086772-01-1, Krotlinger, 1990)
The following supporting studies are available:
28-day oral (gavage) rat study (no guideline, pre-GLP, RL2), NOAEL (male) = 300 mg/kg bw/day, NOAEL (female) = 100 mg/kg bw/day (M- 087319-01-1, Flucke, 1978)
90-day oral (dietary) rat study (no guideline, pre-GLP, RL2), NOAEL = 3.6 mg/kg bw/day (M- 087254-01-1, Vogel, 1983, M-086368-01-1, Krotlinger, 1984)
90-day oral (dietary) dog study (no guideline, pre-GLP, RL2), NOAEL = 2.68 mg/kg bw/day (M- 087344-01-1, Hoffmann, 1980)
21-day dermal rabbit study (no guideline, pre-GLP, RL2), NOAEL = 50 mg/kg bw/day (M- 087328-01-1, Flucke, 1978)
28-day inhalation rat study (no guideline, pre-GLP, RL3), NOAEC = 94.3 mg/m³ (M- 087340-01-1, Thyssen, 1979)

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

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

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.

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.

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

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

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

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.

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.

Key result

Dose descriptor:

NOAEL

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

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

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

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

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.

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)

Reference 2

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 1980 to May 1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

EPA OPP 83-1 (Chronic Toxicity)

Version / remarks:

1984

Deviations:

no

Principles of method if other than guideline:

In a chronic toxicity study on dogs, groups of six male and six female beagles were administered Triflumuron in the diet for a period of 12 months. The concentrations employed were 0, 40, 200 and 1000 ppm equivalent to an average total test compound intake of 0, 13.95, 69.02 and 342.18 mg/kg bw/day for the combined sex. The examinations included daily checks for mortality and
appearance/behaviour, daily food consumption, monitoring of water consumption, weekly body weight determinations and reflex tests, pulse rate, body temperature measurements and ophthalmoscopic examinations on six occasions during the course of the study as well as hematology, clinical chemistry, urinalysis, gross necropsy examinations and histopathology.

GLP compliance:

no

Remarks:

The study was performed before GLP principles were in place.

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

Prior to the start of the study, all dogs were dewormed and were vaccinated against distemper, contagious canine hepatitis, and leptospirosis. Temporary therapeutic and prophylactic measures were taken as indicated by standard veterinary practice.

After an acclimation period, the dogs for the study were selected, classified according to sex and body weight, and then randomly apportioned into groups composed of 6 males and 6 females each. At the time of the random formation of the groups (Week -1) , the dogs were 20-31 weeks old and weighed 6.4 - 10.6 kg.

During the study, all males and females were housed in individual cages in two kennels. The temperature in both kennels was between 20 - 22°C. The mean humidity was about 50%. Brief deviations in the ambient conditions (temperature, humidity), which could occur in connection with the hosing down of the kennels and cages, had no clinically perceptible influence on the state of health of the experimental animals. The kennels were illuminated by diffuse light (skylight), but primarily by fluorescent lights that automatically regulated the 12-hour day /night cycle. Except for weekends and holidays, all dogs, separated by sex, were regularly exercised every morning for about 30 minutes in a space specifically for this purpose directly adjacent to the kennels. During the daily exercise period, the cages and kennels were hosed down with ample amounts of warm water. On workdays, the cages were again hosed down in the afternoon. Prior to the initial placement of the dogs in the kennels and during Weeks 6, 13, 26, and 39, the cages and kennels were thoroughly cleaned out with warm water under high pressure and subsequently disinfected with disinfection solution (approx. 5% Gevisol). During Week -1, Kennel 8 was additionally disinfected with Rapidosept solution.

The dogs were returned to their cages only after the disinfectant had been allowed to act for about 4 hours and thorough hosing had then ensured that all disinfectant had been completely removed. The dogs of all groups received the standard feed [complete feed for dogs, ground twice]. This pulverized dry feed, with which the test compound was uniformly mixed at the respective concentration per test substance group, was given to all dogs at the following daily amounts: 300 g from Week 1 - 13; 330 g from Week 14 - 20; 350 g from Week 21 - 26; 380 g from Week 27 - 38; and 400 g from Week 39 - 52.

Route of administration:

oral: feed

Details on route of administration:

The feed/test compound mixtures were prepared in an MGT mixing granulator (drum capacity: approx. 40 liters).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test item in the animal feed was determined at five different times during the study. The homogeneity was determined once.
The stability of the test item in the feed was determined during a storage period of 10, 11 and 14 days.
Analyses were done using a validated method.
Prior to the start of the study, it had been demonstrated that the test compound was stable in the dry feed for a minimum of 11 days, that it was stable in the feed paste for a minimum of 24 hours, and that it was homogeneously distributed in the mixture.

Duration of treatment / exposure:

The test substance was administered daily to the dogs for a period of 12 months.

Frequency of treatment:

Continuous exposure via feed.

Dose / conc.:

40 ppm

Remarks:

Low dose group, equivalent to 13.95 mg/kg bw/day males/females

Dose / conc.:

200 ppm

Remarks:

Mid dose group, equivalent to 69.02 mg/kg bw/day males/females

Dose / conc.:

1 000 ppm

Remarks:

High dose group, equivalent to 342.18 mg/kg bw/day males/females

No. of animals per sex per dose:

6

Control animals:

yes

Observations and examinations performed and frequency:

General examinations: The appearance and behavior of all animals were observed several times each day during feeding, care, cleaning of the kennel, and the exercise period. The time taken for consumption of the feed was also observed as was the number of times the water bowls needed filling in order to determine whether group-related differences existed.

Anomalies were recorded in the study log. Daily feed consumption and weekly body weights (basically every 7 days) were determined individually for each dog of the reflexes of each animal (pupillary reflex, corneal reflex, patellar reflex, flexor reflex, and extensor thrust reflex) were tested prior to the start of the study (Week -1) and during Weeks 6, 13, 26, 39, and 52. At these same times, body temperature was measured with an electronic, clinical thermometer and pulse rate was taken at the femoral artery. Since various dogs had shown common cold symptoms (coughing) , body temperature was also taken during Week 20 (unscheduled).

Sacrifice and pathology:

Gross pathology and Histological technique:
At the end of the study, all dogs were sacrificed by exsanguination under deep Evipan narcosis, necropsied, and grossly examined. The brain, heart, testes, liver, lungs, spleen, adrenals, kidneys, ovaries, pancreas, prostate, thyroid, and thymus were weighed. Two bone marrow smears were prepared per dog. The bones were decalcified in EDTA. The tissues fixed in 4% formaldehyde (brain) or Bouin's fluid were embedded in Paraplast, from which approx. 5 urn sections were obtained. The sections were stained with hematoxylin and eosin (H&E), periodic acid-Schiff stain (PAS), and/or with Turnbull's blue (Fe). In addition, frozen liver sections approx. 15 pm thick (fixed in 10% formaldehyde) were stained with oil red 0 (ORO) for detection of fat. Bone marrow smears were stained by the method of Pappenheim (MGG) [May-Gruenwald-Giemsa stain].

Other examinations:

Ophthalmoscopy: The eyes of all animals were directly examined using a ophthalmoscope
prior to the start of the study (Week -2) and during Weeks 6, 13, 26, 39, and 52. For documentation, the fundi were photographed prior to the start of the study and during the final examination using a special fundus camera. In the ophthalmoscopic examination, the external parts of the eye (conjunctivae, eyelids, sclera, cornea) were inspected first. In addition to the fundus, the transparent media (cornea, anterior chamber, lens, vitreous body) were evaluated with the aid of the ophthalmoscope.

Clinical signs:

no effects observed

Description (incidence and severity):

The dogs displayed a predominantly normal nutritional state during the entire treatment period. Those dogs that deviated from normal and were evaluated as normal-to-thin or thin and normal-to-fat or fat were distributed about equally among all groups. Thus, treatment at concentrations up to and including 1000 ppm had no remarkable influence on the nutritional state of the experimental animals.

The animals of none of the groups differed from the others in appearance and behavior. All dogs were active, clinically unremarkable, and displayed smooth, well-groomed hair coats. On the whole, vomiting was observed only very sporadically for a few dogs of all groups, and there was no group-related (and thus treatment related) increased incidence of this finding.

There was also no group-specific increased incidence of the findings with respect to the nature of the feces. Occasional (a total of 2 x), minimal amounts of blood found in the feces of one control dog are attributed to parasites in the intestinal tract, but not to treatment with the test compound.

Limited therapeutic measures, regarded as necessary because of the constant and intensive monitoring of all dogs, were distributed among the dogs of all groups, and there was no group-related increased incidence. Such treatments were temporary and were made only on a symptomatic basis. All dogs were prophylactically dewormed during Weeks 4, 26, and 39; because of common cold symptoms, each dog was also given a single administration of 2 mL Tardomyocel during Week 20 (Kennel 8) or Week 21 (Kennel 39). The above-mentioned therapeutic or prophylactic measures had no detectable influence on the results of treatment.

Mortality:

no mortality observed

Description (incidence):

The 12-month administration at concentrations up to and including 1000 ppm was survived by all dogs.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no group-related differences. It was concluded that no treatment-related influence on the body weights was found.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No treatment-related reduction in feed consumption was found. With only a few exceptions unrelated to dose, the dogs consistently consumed the entire amount of their ration. The results are attached below in tabular form.

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

no effects observed

Description (incidence and severity):

No difference was observed between the dogs of all groups with respect to consumption of drinking water.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The ophthalmoscopic examinations, performed at regular intervals on all dogs, showed no treatment-related changes in the transparent media (cornea, anterior chamber, lens, vitreous body) or in the fundus.
Clinical observation revealed no impairment of the eyesight of any of the dogs.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The results are attached below in tabular form.
40 ppm: Tendency towards dose-related increase of reticulocytes.
200 ppm: Haemoglobin and erythrocytes were reduced. MCV increased and MCH decreased. In compensation of the anaemic damage, reticulocytes and thrombocytes increased. Howell-Jolly bodies and Heinz bodies increased. Target cells were seen from week 39 onwards and occasionally
polychromatic and acidophilic normoblasts as well as anulocytes.
1000 ppm: Haemoglobin, haematocrit and erythrocytes were reduced. MCV increased and MCH decreased. In compensation of the anaemic damage, reticulocytes and thrombocytes increased. Howell-Jolly bodies and Heinz bodies increased. Target cells were seen from week 39 onwards and occasionally polychromatic and acidophilic normoblasts as well as anulocytes. in some dogs, pronounces polychromatophilia, anisocytosis and poikilocytosis were found.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The results are attached below in tabular form.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The urinalyses provided no indication of an influence of test item on the composition of the urine at concentrations up to and including 1000 ppm. The results are attached below in tabular form.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Comparison of the absolute and relative organ weights showed no remarkable differences between the dogs of the test item treated groups and the control group for the following organs: heart, lungs, kidneys, testes, ovaries, thyroid, adrenals, thymus, prostate, brain, and pancreas.
The mean liver weight, both absolute and relative, of the high dose group males was greater than control, but the difference was not statistically significant. There was no similar difference for the females.
Statistically significant differences from control were determined for the spleen weight of the dogs of the mid and the high dose group. Both the absolute and the relative spleen weights of the high dose group dogs were substantially greater than control. The difference was less marked for the dogs of the mid dose group. The mean spleen weight of the dogs in the low dose group was also greater than control, but the difference was not statistically significant. The results are attached below in tabular form.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The following changes are considered to be related to the treatment. Gray-blue discoloration of the renal capsule in one animal of the 200 ppm group and in 4 animals of the 1000 ppm group. Green-brown or brown discoloration of the renal cortex in 2 animals of the 200 ppm group and in 6 animals of the 1000 ppm group. Dark urine in one animal of the 200 ppm group and in 5 animals of the 1000 ppm group. Red-brown discoloration of the distal end of the femur in one animal of the 200 ppm group and in 2 animals of the 1000 ppm group. Dark discoloration of the bone marrow in 2 animals of the 1000 ppm group. Furthermore, while severe congestion and dark discoloration of the spleen was observed in only 2 control animals, this was the case in 4 animals of the 40 ppm group, in 6 animals of the 200 ppm group, and in 10 animals of the 1000 ppm group. An increase in pigment deposits in the liver, kidneys, and/or spleen was observed in animals of all treatment groups.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological findings:

Liver: Starting at 40 ppm, mild to moderate deposits of FE-positive pigment were observed in the sinus (phagocytosed by RES cells) and, in some cases, in the hepatocytes. Starting at 200 ppm, brown, FE-negative and ORO-negative pigment was also observed in the sinus endothelial cells.

Kidneys: Starting at 200 ppm, mild to moderate deposits of FE-positive pigment were observed in the plasma of epithelial cells of the proximal convoluted tubules. Starting at 40 ppm, brown, FE-negative and PAS-negative pigment was observed in the same location.

Spleen: Starting at 40 ppm, increased congestion, increased deposits of FE-positive pigment, and/or increased erythropoiesis were observed.

Bone marrow: All animals of the 1000 ppm and 200 ppm groups had a reactive, hypercellular bone marrow with increased erythropoiesis and fewer fat cells. All other histopathological findings are considered random occurrences in individual animals and are not considered
to be induced by treatment. The results are attached below in tabular form.

Other effects:

no effects observed

Description (incidence and severity):

Body temperatures did not differ remarkably between the dogs of any groups. This was equally true for pulse rates.

Key result

Dose descriptor:

LOAEL

Effect level:

13.95 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

histopathology: non-neoplastic

Key result

Dose descriptor:

LOAEL

Effect level:

40 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.:

40 ppm

System:

haematopoietic

Organ:

blood

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Following marginal blood damage even at 40 ppm triflumuron, and a dose-related increase in spleen weight, it was established that no dose level was found in this study which was tolerated by dogs for 12 months without damage. 

 

 

Conclusions:

A 12-month dog study was performed using dietary concentrations of 40, 1200 and 1000 ppm. A NOAEL was not identified due to findings consistent with haemolytic anaemia at all dose levels.

Executive summary:

In a chronic toxicity study, triflumuron was administered in the diet to groups of beagle dogs each consisting of 6 males and 6 females for 12 months at concentrations of 0 ppm, 40 ppm, 200 ppm and 1000 ppm. Treatment at concentrations up to and including 1000 ppm was survived by all dogs and resulted in no changes in their appearance or behaviour. The consumption of feed and water was not affected. There were no differences between treated and control dogs with regard to body temperature, pulse rate, or the findings of neurological and ophthalmoscopic examination. Concentrations up to and including 1000 ppm had no influence on the development of body weight. The nutritional state of the dogs was predominantly normal. There were signs of test compound-related damage to the red blood cells. Reduced erythrocyte counts, reduced haemoglobin and MCHC values indicate hypochromic anaemia at dose levels of 200 ppm and higher. At 40 ppm and higher, there was a dose-related increase in reticulocyte counts. At 200 ppm and higher, histological examination revealed a reactive, hypercellular bone marrow and foci of extramedullary erythropoiesis in the spleen.  Bone marrow of dogs at 200 and 1000 ppm showed an increase in the thrombocyte count.  Increased pigment deposits were found in the liver, kidneys, and spleen of the dogs of all treated groups. At dose levels of 40 ppm and higher, the mean spleen weight was higher than control. At 200 ppm and 1000 ppm, this difference was statistically significant. Methaemoglobin levels at 1000 ppm were significantly higher than controls. Blood coagulation was not affected by treatment. There were no indications of any primary liver damage. The slightly higher mean liver weight of the dogs at 1000 ppm in comparison to controls, and the dose-related increase in bilirubin concentration in the plasma at 200 ppm and higher are interpreted as secondary findings resulting from primary blood damage.  On the basis of the findings of clinical chemistry, haematology, urinalyses, gross examination of the kidneys, and the comparison of kidney weights, there were no indications of damage to the kidneys. On the basis of the clinical observations, the laboratory findings, and the gross and histopathological examinations, there were no indications of damage to any other organ systems. Taking account of marginal blood damage at 40 ppm, a clear NOAEL could not be determined for this study.

 

Reference 3

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 1982 - January 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

EPA OPP 83-1 (Chronic Toxicity)

Deviations:

no

Principles of method if other than guideline:

In a supplementary chronic toxicity study on dogs, groups of six male and female beagles were administered Triflumuron in the diet for a period of 12 months. The concentrations employed were 0 and 20 ppm equivalent equivalent to 0.72 mg/kg bw/day for the combined sex. The examinations included daily checks for mortality and appearance/behaviour, daily food consumption, monitoring of water consumption, weekly body weight determinations and reflex tests, pulse rate, body temperature measurements and ophthalmoscopic examinations on six
occasions during the course of the study as well as hematology, clinical chemistry, urinalysis, gross necropsy examinations and histopathology.

GLP compliance:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

Purebred beagle dogs, which had been selected from a group of 36 males and 36 females, were used for the study. During the quarantine period, all dogs were revaccinated with Candur SHL against distemper, contagious canine hepatitis, and leptospirosis. One dog was treated with Leukomycin eye ointment for purulent conjunctivitis. During the acclimation period, occasional cases of inflammation of the palpebral conjunctivae or partial corneal opacity were treated with Leukomycin eye ointment and Scheroson -F ophthalmicum, respectively. All dogs were dewormed twice with Uvilon (220 mg/kg body weight) prior to the start of the study.

At the end of the acclimation period, the dogs for the study were selected, classified according to sex and body weight, and then randomly apportioned into groups composed of 6 males and 6 females each. At the time of the random formation of the groups (Week -1), the dogs were 20 - 27 weeks old and weighed 7.2 - 10.3 kg. The resulting makeup of the various groups is presented in the randomization list. The dogs were identified by means of ear tattoos and numbered collar tags.

The dogs of all groups received complete feed for dogs, ground twice. This pulverized dry feed, with which the test compound was uniformly mixed at the respective concentration group, was given to each dog at the following daily amounts: 300 g from Week 1-5; 330 g from Week 6-8; 380 g from Week 9 - 21; 400 g from Week 22 - 26; and 430 g from Week 27 to the end of the study.

Immediately prior to administration to the dogs, the pulverized feed (control group) and the feed/test compound mixture (group I) were mixed 1:1 with lukewarm tap water. Using a HOBART mixer, a homogeneous feed paste was prepared from the feed/water mixture with a powered kneading blade. This feed paste was regularly placed in the cages of all dogs every morning, normally between 10:00 a.m. and noon, so that it was available to the dogs for up to 20 hours in case of delayed feeding. The feed unconsumed up to the next feeding was weighed back in order to determine individually the amount of feed consumed and thus the amount of test compound ingested. Tap water in bowls was available to the dogs ad libitum during the entire study.

Route of administration:

oral: feed

Details on route of administration:

The test substance was uniformly mixed with the feed (Ssniff HH) at a concentration of 20 ppm for a period of 12 months.

Details on oral exposure:

The test substance was administered via feed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The feed/test compound mixture was prepared in an MGT mixing granulator (drum capacity: approx. 40 liters).
Prior to the start of the study, it had been demonstrated that the test compound was stable in the dry feed for a minimum of 10 days, that it was stable in the feed paste for a minimum of 24 hours, and that it was homogeneously distributed in the mixture.
Analytics were done using a validated method.
The concentration of the test item in the animal feed was determined at five different times during the study (Weeks 1, 13, 26, 39 and 52).

Duration of treatment / exposure:

12 months

Dose / conc.:

20 ppm

Remarks:

Equivalent to 0.72 mg/kg bw/day for the combined sex

No. of animals per sex per dose:

6

Control animals:

yes

Observations and examinations performed and frequency:

The appearance and behavior of the dogs were observed several times each day during feeding, care, cleaning of the kennels, and the exercise period. The time taken for consumption of the feed was also observed, as was the thirst of the dogs as measured by the number of times the water bowls needed filling. Deviations from the physiological norm and/or between the dogs of the control group and those of group I were recorded. Daily feed consumption and weekly body weights (basically every 7 days) were determined individually for each dog. Individual nutritional state was evaluated at least once per month.

The reflexes of each animal (pupillary reflex, corneal reflex, patellar reflex, flexor reflex, and extensor thrust reflex) were tested prior to the start of the study (Week -2) and during Weeks 6, 13, 26, 39, and 52. At these same times, body temperature was measured rectally with an electronic, clinical thermometer and pulse rate was taken at the femoral artery.

The eyes of all dogs were directly examined using a bifocal ophthalmoscope prior to the start of the study (Week -2) and during Weeks 5, 13, 29, 39, and 52. In the ophthalmoscopic examination, the external parts of the eye (conjunctivae, eyelids, sclera, cornea) were inspected first. In addition
to the fundus, the transparent media (cornea, anterior chamber, lens, vitreous body) were evaluated with the aid of the ophthalmoscope.

Hematological tests, clinical chemistry tests, and urinalyses were performed on all dogs prior to the start of the study (Week -2) and during Weeks 6, 13, 26, 39, and 52.

Hematology parameters: Hemoglobin, hematocrit, erythrocyte and leukocyte counts, mean corpuscular haemoglobin, mean corpuscular volume of the individual erythrocytes, mean corpuscular hemoglobin concentration, thrombocyte count, reticulocyte count, thromboplastin, blood sedimentation rate, leukocyte differential count.

Clinical chemistry: Blood glucose, plasma urea, plasma creatinine, bilirubin in the plasma, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, total protein, cholesterol, glutamate dehydrogenase, serum protein, sodium, potassium, calcium, chloride, methemoglobin.

For the collection of urine samples, all animals were placed in individual metabolism cages from approx. 8 a.m. to 2 p.m. Feed and water were withheld during this time. Prior to placement in the urine collection cages, each animal was administered approx. 250 mL tap water by means of
an oral intubation tube.

Urinalysis parameters: Volume, specific gravity, protein, glucose, blood, bilirubin, ketone bodies, and pH.

Sacrifice and pathology:

All dogs of the study were sacrificed by exsanguination under deep Evipan narcosis, necropsied, and grossly examined.
The heart, lungs, liver, kidneys, spleen, thyroid, adrenals, prostate, brain, pancreas, and testes or ovaries were weighed.
Bone marrow smears were stained with May-Gruenwald-Giemsa stain (MGG).
Histopathological examination was performed on Heart, lungs, liver, spleen, kidneys, brain, adrenals (2 x), thyroid, pituitary, testes (2 x), epididymides (2 x), prostate, ovaries (2 x), uterus (2 x), parotid, esophagus, stomach (2 x), intestines (4 x), pancreas, gall bladder, skeletal musculature, urinary bladder, aorta, lymph nodes, thymus, mammary glands, eyes, optic nerves (2 x), sciatic nerve, and bone and bone marrow (femur).

Other examinations:

Ophthalmoscopy: The eyes of all dogs were directly examined.

Clinical signs:

no effects observed

Description (incidence and severity):

There was no difference in appearance and behavior between the dogs administered 20 ppm and the control dogs. The nutritional state was predominantly normal throughout the entire study. At the end of treatment, a few dogs of both groups were assessed as normal-to-fat.

The monitoring of body temperature and of pulse rate prior to the start of the study, at the intermediate examination times, and at the final examination revealed no remarkable differences between the treated dogs treated and controls either for body temperature or for pulse rate.

Mortality:

no mortality observed

Description (incidence):

All dogs survived until scheduled necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no remarkable differences in mean body weight between the dogs of the control group and those of the dose group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No difference was found between the feed consumption of exposed and control animals.
Based on the amounts of test compound administered with the feed, the amounts of test compound ingested by the dogs treated at 20 ppm were calculated to be approx. 2.84 g/dog total and approx. 54.7 mg/dog per week. An overview of the results is included in Tables 5 to 8, "Overall remarks, attachments section")

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

no effects observed

Description (incidence and severity):

No difference was found between the water intake of exposed and control animals.

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

The erythrocyte count, haemoglobin concentration, hematocrit, leukocyte, and reticulocyte counts were within the range of normal variation for all dogs at all examination times. The same followed for the thrombocyte count and coagulation. An overview of the results is included in Tables 9 and 10, "Overall remarks, attachments section")

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no remarkable differences between the values for blood glucose of the dogs of the dose group and control. The values for urea and creatinine measured in the plasma also provided no group-specific differences and gave no indication of damage to the kidneys. With regard to the total protein and cholesterol concentrations in the plasma, there were also no remarkable differences between the animals of the two groups. The same is true of the mean activity of the GOT, GPT, AP, and GLDH. The activities measured for the GPT and GLDH for one dog were outside the range of normal variation. This dog was clinically unremarkable, and gross pathological and histopathological examination also failed to reveal any morphological changes in the liver. Any relation to treatment with the test compound can be ruled out, since this was a control dog.

The serum electrolytes sodium, potassium, calcium, and chloride were also within the range of normal variation at all examination times and revealed no differences between the dogs treated with the test substance and control. Overall, the electrophoretic separation of the serum revealed no group related differences and thus no indication of deviations from the norm that could be related to treatment with the test compound. An overview of the results is included in Tables 11 and 12, "Overall remarks, attachments section")

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Blood was sporadically detected in the urine of a total of 5 dogs. Repeat tests a few days after the positive blood finding or after the conclusion of estrus consistently showed that the urine was free of blood for all of the dogs noted above. An overview of the results is included in Tables 1 to 4, "Overall remarks, attachments section")

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The testing of the reflexes at the examination times (pupillary reflex, corneal reflex, patellar reflex, flexor reflex, and extensor thrust reflex) did not provide any reactions deviating from the physiological norm for any of the dogs.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The comparison of the absolute and relative organ weights revealed no remarkable differences between the dogs of the dose group and the control group following treatment. An overview of the results is included in Table 13, "Overall remarks, attachments section")

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No group-specific histopathological findings were obtained. Iron-positive pigments in the liver, spleen, and kidneys of the dogs treated were not increased in comparison with control. An overview of the results is included in Tables 14 to 23, "Overall remarks, attachments section")

Key result

Dose descriptor:

NOAEL

Effect level:

0.72 other: mg/kg bw day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Effect level:

20 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

On the basis of all results of clinical observation, the laboratory findings, and the results of gross pathological and histopathological examination, 20 ppm was established as the no observable effect level for administration to dogs for a 12-month period.

The mean analysed concentrations were 19±8.1 mg/kg feed (nominal: 20 mg/kg feed).

The feed was concluded to be homogeneous (mean concentration was 95% of the nominal concentrations). 

The test substance was stable in the dry feed mixture over a period of 10 days, taking into consideration the tolerance range of ± 20% relative to
the nominal concentration (average 84%).

 

Conclusions:

This supplementary 12-month dog study was performed using a single dietary concentration of 20 ppm. No treatment-related findings are reported.

Executive summary:

As a supplement to a chronic toxicity study of on dogs, in which no observable effect level was not found because of marginal damage to the blood at 40 ppm, a study was conducted in which triflumuron was administered in the feed ration to a group of beagle dogs consisting of 6 males and 6 females for one year at a concentration of 20 ppm. The untreated control group consisted of 6 male and 6 female beagle dogs. In the current study, the dose of 20 ppm was tolerated without effects every respect (appearance, behaviour, feed consumption, body weight, ophthalmoscopy, pulse rate, and reflexes). No differences whatsoever were found between the triflumuron group and control that could be related to treatment. There were no indications of damage to the blood following the chronic administration of 20 ppm. There was no evidence of any influence on the liver caused by 20 ppm. There were no indications of treatment-related damage to the kidneys. On the basis of all results of clinical observation, the laboratory findings, and the results of gross pathological and histopathological examination, 20 ppm was established as the no observable effect level for administration to dogs for a 12-month period.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1.42 mg/kg bw/day

Study duration:

chronic

Species:

dog

Quality of whole database:

A comprehensive and high-quality database of repeated oral toxicity studies of up to chronic duration are available for the rat, mouse and dog. The available information comprises adequate, reliable (Klimisch score 2) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VII and VIII of Regulation (EC) No 1907/2006.

System:

haematopoietic

Organ:

blood

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

October 1987 - November 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Version / remarks:

1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on species / strain selection:

HC:NZW

Sex:

male/female

Details on test animals or test system and environmental conditions:

Only healthy, symptom-free animals were used for the study. At the start of the study, the male animals exhibited a mean initial weight of 2.92 (2.64 - 3.23) kg, and the females of 2.75 (2.31 - 3.10) kg. Based on these body weights, the age of the animals was around 13 and 11 weeks, respectively.
Animal housing: During the adaptation and study periods, the animals were individually kept under conventional conditions in Cellidor rabbit cages. Following a study duration of three weeks in standard metal cages. The stool trays located beneath the cages were changed twice weekly. During the six-hour exposure periods, the animals were confined in restraining racks. These prevented the formulation from being rubbed off and orally ingested. All animals used in this study were housed in one animal room during the adaptation, study and post-exposure observation periods. The room was changed in the fourth week of the study for procedural reasons.

Room climate conditions:
The room climate was set as follows.
Room temperature : 22 ± 2° C
Humidity (relative) : about 50%
Light/dark cycles : 12 hours; artificial lighting from 6.00 to 18.00 hours Central European Time
Air exchanges : About 10 times per hour.

Occasional deviations from these standards took place, for example due to cleaning of the animal room. They had no apparent effect on the course of the study.

Feeding: The food consisted of "Ssniff K Sole Diet for Rabbits" produced by Flange Kraftfutterwerk GmbH in Soest, and of tap water from watering bottles. Water was available for unlimited consumption. The food was provided to the animals each day from automatic rabbit food dispensers.

Type of coverage:

semiocclusive

Vehicle:

physiological saline

Remarks:

The test substance was formulated with 2 % v/v Cremophor EL in sterile physiological saline solution prior to each treatment.

Details on exposure:

The fur of the back and flanks of all rabbits was shaved off 48 hours before initiating treatment. The hair which grew back during the study was shaved off the skin areas marked for treatment twice weekly. Once daily on 15 consecutive working days (five times per week), the test substance formulations were applied to four-ply gauze patches with syringes; the patches were fixed to the dorsal and flank skin of the experimental animals using Fixomull-stretch, and allowed to remain there for a period of six hours. The application volume for each concentration was 2 ml/kg body weight. The treatment area measured 11 x 12 cm. The treated skin areas were cleaned with soap and water at the conclusion of each exposure. The rabbits were confined in restraining racks and additionally immobilized by a rubber sleeve during the exposure period to keep the occlusive dressings tight and prevent the animals from gnawing on them. No food or water were provided to the rabbits during this time. This treatment method is used to determine the local and systemic tolerance of the test substance following repeated dermal exposure to estimate the risk to humans in cases of repeated dermal contact with the test substance.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity and stability of the test substance in the treatment medium were examined before initiating the study. The concentration of the test item was verified analytically.

Duration of treatment / exposure:

The test substance was applied once daily on 15 consecutive working days (five times per week).

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

5;
Five additional animals per sex were used to monitor the reversibility of the treatment (two-week recovery phase after concluding the application) in the control and the highest dose groups.

Control animals:

yes, concurrent vehicle

Details on study design:

The post-observation period was 2 weeks.

Observations and examinations performed and frequency:

General examinations: The appearance and behavior of the animals were monitored at least once daily during the study and post-exposure observation periods.

Food consumption: The food intakes of the individual animals were determined once weekly from the difference in the weight of food provided and that of the unconsumed food. The mean individual food intakes and their standard deviations were calculated for each group from these primary food intake data.

Body weights: The body weights of the animals were determined by weighing them before initiating the study and at the start of each study week.

Testing for local skin tolerance: The treated skin areas were inspected for inflammation reactions (redness and swelling) before initiating the study and 24 hours after each treatment.

Laboratory tests: Laboratory tests were performed on blood samples taken from all animals before initiating treatment, and samples taken from all survivors following the three-week treatment period (24 hours after the last exposure) and at the end of the two-week post-exposure observation period.

Hematological examinations: The following specific tests were performed. Erythrocyte and leucocyte counts, reticulocyte count, Heinz inclusion bodies, hemoglobin, hematocrit, thrombocyte count, mean corpuscular erythrocyte volume, mean cell hemoglobin content of erythrocytes.

Sacrifice and pathology:

48 to 72 hours after the last treatment, and at the end of the two-week post-exposure observation period, the appropriate groups of rabbits were sacrificed by exsanguination under deep anesthesia.

Other examinations:

Clinical chemistry:

Blood: Aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase, urea, blood sugar, creatinine, bilirupin, DPD method, total protein, inorganic phosphate, sodium, potassium and calcium, chloride.

Liver tissue: N-demethylase, O-demethylase, cytochrome P-450, Triglycerides.

Organ weights: Organs of sacrificed animals were weighed.

Statistics:

The arithmetic group means, standard deviations, and upper and lower confidence limits at confidence levels were calculated from the body weights, dermal findings, medical laboratory tests and organ weights, and the test cohort results compared with those for the control cohort using the significance test (two-tailed U test) of Mann and Whitney and Wilcoxon at significance levels of a = 5 % and a = 1 %. Significant differences relative to the control group are marked with a "+" for p < 0.05 and with "++" for p < 0.01 in the mean figure tables located in the results section.

Clinical signs:

no effects observed

Description (incidence and severity):

The appearance, behaviour and mortality were unaffected in all dose groups. No delayed effects were observed.

Dermal irritation:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight development was unaffected in all dose groups.

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Several hematological parameters - such as the erythrocyte count, the hematocrit and hemoglobin values, and the reticulocyte count in male animals; and the MCHC value in the females - differed from the control figures to a statistically significant extent at the start of the study. Since these differences were distributed throughout various groups, were not large, and also lay within the reference range, they represent normal biological scattering, and no significance is attached to them.

In the 1000 mg/kg b.w. group, the erythrocyte and reticulocyte counts as well as the hematocrit and hemoglobin values (both sexes), and the MCHC value (males only) differed from those of the controls to a statistically significant extent at the end of the treatment period. The reticulocyte counts were higher and the values of the other parameters lower than in the controls. The elevated Heinz body count in the males of this group is due to the high figure obtained for a male. In the 100 mg/kg b.w. group, the erythrocyte counts as well as the hematocrit and hemoglobin values were depressed in male animals and elevated in females, and the thrombocyte counts were depressed (females only); the deviations were independent of the dose but statistically significant.

All other values are within the reference range for this strain of animals except for the reticulocyte count and hemoglobin concentration parameters, where several results lay outside the range. The differential blood count in the dose groups underwent no treatment-related effect relative to the controls. One normoblast was found in each of two 1000 mg/kg b.w. group males, one at the start of the study, and one after 21 days. The hematology test results are compiled as mean figures in the Tables 4 and 5, "Overall remarks, attachments section".

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The results determined for the dose groups at the onset of the study exhibited no significant differences relative to the controls in any of the parameters examined. At the end of the treatment period, the test results for male animals showed no significant differences relative to the controls.

The ALAT activities were higher in the 1000 mg/kg b.w. females. Except in two animals, the individual values lay within the reference range. ALAT activities of both animals lay at the upper limit of the 2a range even before the study was initiated. However, no toxicological significance is thus attached to these elevated figures, especially since no further correlatives for hepatic damage were present.

The N- and O-demethylase activities, the cytochrome P-450 level and the content of triglycerides in the liver tissue were examined. The results show that no significant induction of the microsomal enzyme systems examined resulted from the treatment, and no biologically relevant differences between the treated and control animals were present.

In addition, no toxicological significance is attached to the elevated concentrations of the P-450 system.

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Distinctly swollen spleen was observed at 300 and 1000 mg/kg bw.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

300 mg/kg bw: engorgement and pronounced hemosiderosis in the spleen
1000 mg/kg bw: engorgement and pronounced hemosiderosis in the spleen
The effect was reversible after the 14 days post-treatment observation period.

Other effects:

no effects observed

Description (incidence and severity):

Macroscopic assessment of the skin in the vicinity of the treatment area showed no redness in the animals of any group. The skinfold measurement results similarly showed no significant alterations in any of the groups. The treatment area was partially squamous in one animal.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day

System:

haematopoietic

Organ:

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

Based on the results of a 3-week dermal rabbit study at dose levels of 100, 300 and 1000 mg/kg bw/d, the NOAEL was concluded to be 100 mg/kg bw/d, based findings consistent with haemolysis at higher dose levels.

Executive summary:

The local and systemic toleration of the test substance triflumuron by rabbits with intact skin was tested in a three-week subacute dermal toxicity study with a two-week recovery period. The test substance was formulated with 2 % v/v Cremophor EL in sterile physiological saline solution, and doses as specified below in an application volume of 2 ml/kg body weight applied to the shorn dorsal and flank skin on each of 15 consecutive working days (five days per week); the dose was then allowed to remain in contact with the skin for a period of six hours. The dose groups were 0, 100, 300 and 1000 mg/kg bw/d. The treatment was tolerated without symptoms by all groups. In none of the dose groups did the body weight development of the animals differ significantly from that of the controls. Neither the clinical chemistry, haematology and organ weight tests which were performed, nor the macroscopic and microscopic assessments of the internal organs afforded any results which could speak for treatment-related effects or damage in the groups exposed to doses up to and including 100 mg/kg bw/d. An effect on the red blood count was observed in the haematology results at 1000 mg/kg bw/d, and was confirmed by splenic alterations. Splenic alterations were also observed at 300 mg/kg bw/d. A slight effect on the red blood count was still apparent in the haematology at the end of the recovery phase, whereas histopathological correlates were no longer observed. No test substance-related local skin reactions were observed. The NOAEL for this study was therefore 100 mg/kg bw/d.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

30

Species:

rabbit

Quality of whole database:

A GLP and guideline study (OECD 410) in rabbits is available.

System:

haematopoietic

Organ:

spleen

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

October 1987 - November 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Version / remarks:

1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on species / strain selection:

HC:NZW

Sex:

male/female

Details on test animals or test system and environmental conditions:

Only healthy, symptom-free animals were used for the study. At the start of the study, the male animals exhibited a mean initial weight of 2.92 (2.64 - 3.23) kg, and the females of 2.75 (2.31 - 3.10) kg. Based on these body weights, the age of the animals was around 13 and 11 weeks, respectively.
Animal housing: During the adaptation and study periods, the animals were individually kept under conventional conditions in Cellidor rabbit cages. Following a study duration of three weeks in standard metal cages. The stool trays located beneath the cages were changed twice weekly. During the six-hour exposure periods, the animals were confined in restraining racks. These prevented the formulation from being rubbed off and orally ingested. All animals used in this study were housed in one animal room during the adaptation, study and post-exposure observation periods. The room was changed in the fourth week of the study for procedural reasons.

Room climate conditions:
The room climate was set as follows.
Room temperature : 22 ± 2° C
Humidity (relative) : about 50%
Light/dark cycles : 12 hours; artificial lighting from 6.00 to 18.00 hours Central European Time
Air exchanges : About 10 times per hour.

Occasional deviations from these standards took place, for example due to cleaning of the animal room. They had no apparent effect on the course of the study.

Feeding: The food consisted of "Ssniff K Sole Diet for Rabbits" produced by Flange Kraftfutterwerk GmbH in Soest, and of tap water from watering bottles. Water was available for unlimited consumption. The food was provided to the animals each day from automatic rabbit food dispensers.

Type of coverage:

semiocclusive

Vehicle:

physiological saline

Remarks:

The test substance was formulated with 2 % v/v Cremophor EL in sterile physiological saline solution prior to each treatment.

Details on exposure:

The fur of the back and flanks of all rabbits was shaved off 48 hours before initiating treatment. The hair which grew back during the study was shaved off the skin areas marked for treatment twice weekly. Once daily on 15 consecutive working days (five times per week), the test substance formulations were applied to four-ply gauze patches with syringes; the patches were fixed to the dorsal and flank skin of the experimental animals using Fixomull-stretch, and allowed to remain there for a period of six hours. The application volume for each concentration was 2 ml/kg body weight. The treatment area measured 11 x 12 cm. The treated skin areas were cleaned with soap and water at the conclusion of each exposure. The rabbits were confined in restraining racks and additionally immobilized by a rubber sleeve during the exposure period to keep the occlusive dressings tight and prevent the animals from gnawing on them. No food or water were provided to the rabbits during this time. This treatment method is used to determine the local and systemic tolerance of the test substance following repeated dermal exposure to estimate the risk to humans in cases of repeated dermal contact with the test substance.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity and stability of the test substance in the treatment medium were examined before initiating the study. The concentration of the test item was verified analytically.

Duration of treatment / exposure:

The test substance was applied once daily on 15 consecutive working days (five times per week).

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

5;
Five additional animals per sex were used to monitor the reversibility of the treatment (two-week recovery phase after concluding the application) in the control and the highest dose groups.

Control animals:

yes, concurrent vehicle

Details on study design:

The post-observation period was 2 weeks.

Observations and examinations performed and frequency:

General examinations: The appearance and behavior of the animals were monitored at least once daily during the study and post-exposure observation periods.

Food consumption: The food intakes of the individual animals were determined once weekly from the difference in the weight of food provided and that of the unconsumed food. The mean individual food intakes and their standard deviations were calculated for each group from these primary food intake data.

Body weights: The body weights of the animals were determined by weighing them before initiating the study and at the start of each study week.

Testing for local skin tolerance: The treated skin areas were inspected for inflammation reactions (redness and swelling) before initiating the study and 24 hours after each treatment.

Laboratory tests: Laboratory tests were performed on blood samples taken from all animals before initiating treatment, and samples taken from all survivors following the three-week treatment period (24 hours after the last exposure) and at the end of the two-week post-exposure observation period.

Hematological examinations: The following specific tests were performed. Erythrocyte and leucocyte counts, reticulocyte count, Heinz inclusion bodies, hemoglobin, hematocrit, thrombocyte count, mean corpuscular erythrocyte volume, mean cell hemoglobin content of erythrocytes.

Sacrifice and pathology:

48 to 72 hours after the last treatment, and at the end of the two-week post-exposure observation period, the appropriate groups of rabbits were sacrificed by exsanguination under deep anesthesia.

Other examinations:

Clinical chemistry:

Blood: Aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase, urea, blood sugar, creatinine, bilirupin, DPD method, total protein, inorganic phosphate, sodium, potassium and calcium, chloride.

Liver tissue: N-demethylase, O-demethylase, cytochrome P-450, Triglycerides.

Organ weights: Organs of sacrificed animals were weighed.

Statistics:

The arithmetic group means, standard deviations, and upper and lower confidence limits at confidence levels were calculated from the body weights, dermal findings, medical laboratory tests and organ weights, and the test cohort results compared with those for the control cohort using the significance test (two-tailed U test) of Mann and Whitney and Wilcoxon at significance levels of a = 5 % and a = 1 %. Significant differences relative to the control group are marked with a "+" for p < 0.05 and with "++" for p < 0.01 in the mean figure tables located in the results section.

Clinical signs:

no effects observed

Description (incidence and severity):

The appearance, behaviour and mortality were unaffected in all dose groups. No delayed effects were observed.

Dermal irritation:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight development was unaffected in all dose groups.

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Several hematological parameters - such as the erythrocyte count, the hematocrit and hemoglobin values, and the reticulocyte count in male animals; and the MCHC value in the females - differed from the control figures to a statistically significant extent at the start of the study. Since these differences were distributed throughout various groups, were not large, and also lay within the reference range, they represent normal biological scattering, and no significance is attached to them.

In the 1000 mg/kg b.w. group, the erythrocyte and reticulocyte counts as well as the hematocrit and hemoglobin values (both sexes), and the MCHC value (males only) differed from those of the controls to a statistically significant extent at the end of the treatment period. The reticulocyte counts were higher and the values of the other parameters lower than in the controls. The elevated Heinz body count in the males of this group is due to the high figure obtained for a male. In the 100 mg/kg b.w. group, the erythrocyte counts as well as the hematocrit and hemoglobin values were depressed in male animals and elevated in females, and the thrombocyte counts were depressed (females only); the deviations were independent of the dose but statistically significant.

All other values are within the reference range for this strain of animals except for the reticulocyte count and hemoglobin concentration parameters, where several results lay outside the range. The differential blood count in the dose groups underwent no treatment-related effect relative to the controls. One normoblast was found in each of two 1000 mg/kg b.w. group males, one at the start of the study, and one after 21 days. The hematology test results are compiled as mean figures in the Tables 4 and 5, "Overall remarks, attachments section".

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The results determined for the dose groups at the onset of the study exhibited no significant differences relative to the controls in any of the parameters examined. At the end of the treatment period, the test results for male animals showed no significant differences relative to the controls.

The ALAT activities were higher in the 1000 mg/kg b.w. females. Except in two animals, the individual values lay within the reference range. ALAT activities of both animals lay at the upper limit of the 2a range even before the study was initiated. However, no toxicological significance is thus attached to these elevated figures, especially since no further correlatives for hepatic damage were present.

The N- and O-demethylase activities, the cytochrome P-450 level and the content of triglycerides in the liver tissue were examined. The results show that no significant induction of the microsomal enzyme systems examined resulted from the treatment, and no biologically relevant differences between the treated and control animals were present.

In addition, no toxicological significance is attached to the elevated concentrations of the P-450 system.

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Distinctly swollen spleen was observed at 300 and 1000 mg/kg bw.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

300 mg/kg bw: engorgement and pronounced hemosiderosis in the spleen
1000 mg/kg bw: engorgement and pronounced hemosiderosis in the spleen
The effect was reversible after the 14 days post-treatment observation period.

Other effects:

no effects observed

Description (incidence and severity):

Macroscopic assessment of the skin in the vicinity of the treatment area showed no redness in the animals of any group. The skinfold measurement results similarly showed no significant alterations in any of the groups. The treatment area was partially squamous in one animal.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day

System:

haematopoietic

Organ:

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

Based on the results of a 3-week dermal rabbit study at dose levels of 100, 300 and 1000 mg/kg bw/d, the NOAEL was concluded to be 100 mg/kg bw/d, based findings consistent with haemolysis at higher dose levels.

Executive summary:

The local and systemic toleration of the test substance triflumuron by rabbits with intact skin was tested in a three-week subacute dermal toxicity study with a two-week recovery period. The test substance was formulated with 2 % v/v Cremophor EL in sterile physiological saline solution, and doses as specified below in an application volume of 2 ml/kg body weight applied to the shorn dorsal and flank skin on each of 15 consecutive working days (five days per week); the dose was then allowed to remain in contact with the skin for a period of six hours. The dose groups were 0, 100, 300 and 1000 mg/kg bw/d. The treatment was tolerated without symptoms by all groups. In none of the dose groups did the body weight development of the animals differ significantly from that of the controls. Neither the clinical chemistry, haematology and organ weight tests which were performed, nor the macroscopic and microscopic assessments of the internal organs afforded any results which could speak for treatment-related effects or damage in the groups exposed to doses up to and including 100 mg/kg bw/d. An effect on the red blood count was observed in the haematology results at 1000 mg/kg bw/d, and was confirmed by splenic alterations. Splenic alterations were also observed at 300 mg/kg bw/d. A slight effect on the red blood count was still apparent in the haematology at the end of the recovery phase, whereas histopathological correlates were no longer observed. No test substance-related local skin reactions were observed. The NOAEL for this study was therefore 100 mg/kg bw/d.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rabbit

Quality of whole database:

A GLP and guideline study (OECD 410) in rabbits is available.

Mode of Action Analysis / Human Relevance Framework

Erythrocyte damage represents the main toxic effect following repeated administration of triflumuron.
Compensation or regeneration processes (highly active bone marrow, extramedullary hematopoiesis in the spleen and frequent appearance of immature erythrocytes in the peripheral blood) were observed as a result of the erythrocyte damage. The elevated metabolic activity of the spleen and the hemosiderosis in spleen, liver and kidneys represent secondary effects. The elevated leukocyte and thrombocyte counts in the peripheral blood sometimes observed simultaneously are viewed in the sense of an entrainment effect of the increased hematopoiesis. The increased hematopoiesis indicates that the primary attack by the substance takes place on the peripheral blood. No impairment in the sense of a hematogenic organ depression or general cytotoxic effect was observed in any study. Elevated liver and kidney weights were repeatedly observed in long-term studies. Since corresponding macroscopic, histopathological or clinical-chemical correlations were absent (exception: single cell liver necroses in the subchronic study on rats), it may be generally assumed that no toxic effect on these organs exists. The elevated organ weights apparently result from the accelerated metabolic and excretion processes which follow erythrocyte damage.

Elevated fluoride levels were determined in the bones and teeth of rats and mice in chronic studies. No macroscopic or microscopic alterations of the bones or teeth were observed. The quantities of fluoride incorporated from Triflumuron were thus insufficient to produce fluorosis even at the highest doses used in the chronic studies and are thus considered as non-adverse.

Considering that the extent of haemolytic anaemia after the administration of triflumuron was not representing a severe functional disorder, it is concluded that the classification with STOT RE Cat. 2 is not justified.

 

Justification for classification or non-classification

Based on the currently available information it is concluded that Triflumuron does not meet the criteria for classification for STOT-RE according to Regulation (EC) No. 1272/2008.