pymetrozine (ISO); (E)-4,5-dihydro-6-methyl-4-(3-pyridylmethyleneamino)-1,2,4-triazin-3(2H)-one

Endpoint summary

Description of key information

- Oral: NOAEL = 100 ppm (both sexes), equivalent to dose levels of 3.12 mg/kg bw/day for males and 3.24 mg/kg bw/day for females, dogs, sub-chronic, 3 months, feeding, OECD 409, Altmann 1992

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Jun 1991 to 12 Sep 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Version / remarks:

Adopted May 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: FIFRA Subdivision F: Hazard Evaluation: Human and Domestic Animals, Paragraph 82-1, EPA office of Pesticide and Toxic Substances of November 1982.

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 34 to 35 weeks (for males), 30 to 34 weeks (for females)
- Weight at study initiation: 9.7 to 12.1 kg (for males), 8.1 to 11.4 kg (for females)
- Fasting period before study: No
- Housing: Two dogs were housed together in a kennel. Each dog was fastened with a chain during the feeding period, thereafter indoor and outdoor runs were available for the dogs. The kennels were provided with heating to give a minimum room temperature of 15°C throughout the year.
- Diet: Certified pelleted standard diet NAFAG 941 Tox, 350 g/animal daily
- Water: ad libitum (tap water)
- Acclimation period: An acclimatisation period of 28 days was allowed between delivery and the start of treatment.
- Other details: Pedigree Beagle dogs, dewormed, vaccinated against rabies, distemper, leptospirosis, canine hepatitis and parvovirus infection.

DETAILS OF FOOD AND WATER QUALITY:
The drinking water quality fulfilled in the critical parameters the specifications of the "Schweizerisches Lebensmittelbuch" (Edition 1972). All batches of diet were assayed for composition and
contaminant levels by the manufacturer.

ENVIRONMENTAL CONDITIONS
- Temperature: Throughout the year a minimum of 15°C
- Photoperiod (hrs dark / hrs light): 12/12
- Other: Music was broadcasted for about 8 hours daily during the lighting phase.

IN-LIFE DATES:
10 Jun 1991 to 12 Sep 1991

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
The appropriate amounts of the test substance were added to pulverised food, mixed, subsequently added to a further portion of pulverised food and mixed homogeneously. To these premixes diet was added to attain the nominal concentrations, blended homogeneously and pelleted.
- Rate of preparation of diet: Three batches of medicated feed were prepared for the 13 weeks test period.
- Mixing appropriate amounts with: Certified pelleted standard diet NAFAG 941 Tox
- Storage temperature of food: The diet was stored in stainless steel containers at room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Samples of the pelleted diet containing the test substance were analysed for concentration, homogeneity and stability of the test substance by an HPLC method. Analysis of homogeneity was performed by analysing samples of each diet from three different segments (beginning, middle, end).

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Once/day, 7 days a week

Dose / conc.:

100 ppm

Remarks:

Group 2. Dietary concentration; equivalent to 3.12 and 3.24 mg/kg bw/day for males and females, respectively.

Dose / conc.:

500 ppm

Remarks:

Group 3. Dietary concentration; equivalent to 13.9 and 14.5 mg/kg bw/day for males and females, respectively.

Dose / conc.:

2 500 ppm

Remarks:

Group 4. Dietary concentration; equivalent to 53.4 and 60.2 mg/kg bw/day for males and females, respectively.

No. of animals per sex per dose:

4

Control animals:

yes, plain diet

Details on study design:

The doses were selected based on the results of a previously conducted range finding toxicity study in Beagle Dogs.
- A low dose of 100 ppm was expected to be a no observable effect level for a 3 month dosing period.
- A dose of 500 ppm was expected to cause slight adverse effects.
- A high dose of 2500 ppm was expected to cause evidence of systemic toxicity without fatalities.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked daily (a.m. and p.m. on working days, a.m. on weekends and holidays), in order to record mortalities and to allow dead or moribund animals to be submitted to
necropsy as soon as possible. In order to detect changes in state of health or behaviour, or any other reactions to treatment, examination was carried out daily.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of all animals was recorded individually at weekly weighing sessions.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food was offered as a constant amount of 350 g/animal/day during a restricted feeding period. Food consumption was measured daily as g/animal/day. Food consumption ratio (FCR) was calculated according to the following formula: FCR = (weekly food consumption in g) / (body weight in kg x 7).

OPHTHALMOSCOPIC EXAMINATION: Yes
After external inspection, the lens, the iris and the fundus were examined with an ophthalmoscope. The appearance of the fundus was documented photographically. Mydriaticum was applied to induce mydriasis. The pupillary reflex was checked in all animals and the third eyelid was examined after local anaesthesia using Novesin 0.4%.
- Time schedule for examinations: Eye examinations were performed in all surviving animals at pretest and week 13.
- Dose groups that were examined: all surviving animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before the exposure, wk 4, 5, 7, 8, 12 and 13. Blood sampling was performed in the morning. Blood was removed from the jugular vein.
- Anaesthetic used for blood collection: not specified
- Animals fasted: Yes, food was withheld for at least 16 hours prior to blood collection.
- How many animals: all surviving animals of each sex and group
- The following parameters were examined:
Red blood parameters: erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, haemoglobin concentration distribution width.
White blood cell parameters: leukocyte count, differential leukocyte count; neutrophils, eosinophils, basophils, lymphocytes, monocytes, large unstained cells.
Blood platelets: Thrombocyte count.
In addition Prothrombin time, Methemoqlobin, Reticulocyte count and Osmotic fragility were measured.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before the exposure, wk 4, 5, 7, 8, 12 and 13. Blood sampling was performed in the morning. Blood was removed from the jugular vein.
- Anaesthetic used for blood collection: not specified
- Animals fasted: Yes, food was withheld for at least 16 hours prior to blood collection.
- How many animals: all surviving animals of each sex and group
- The following parameters were examined: Glucose, urea, creatinine, total bilirubin, direct bilirubin, total protein, albumin, globulins, A/G ratio, cholesterol, phospholipids, sodium, potassium, calcium, chloride, phosphorus inorganic, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, Gamma-glutamyl transpeptidase and creatine kinase.

URINALYSIS: Yes
Chemical urine components, pH and relative density were estimated by an automated urine analyser Clinitek Auto 2000 using solid-phase reagent systems combined with reflectance spectroscopy (chemical constituents and pH), and applying the falling drop method (relative density).
- Time schedule for collection of urine: before the exposure, wk 4, 5, 7, 8, 12 and 13.
- Metabolism cages used for collection of urine: no, urine for analysis was obtained by catheterization
- Animals fasted: Yes, food was withheld for at least 16 hours prior to blood collection.
- The following parameters were examined:
Physical and chemical examination: relative density, pH-value, urine colour, protein, glucose, ketones, bilirubin, blood, urobilinogen.
Microscopic examination: cells, casts, crystals.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At the end of the treatment period and the treatment-free recovery period all surviving controls and treated animals were anaesthetised by intravenous injection of T 61 (Hoechst), exsanguinated and subjected to detailed necropsy.
At autopsy the following weights were recorded from all surviving animals: body (exsanguinated), brain, heart, liver, kidneys, adrenals, thymus, ovaries/testes, spleen and thyroid gland (including parathyroids).
The following organs and tissues were preserved in neutral buffered 4% formalin: skin, mammary area, spleen, cervical lymph node, mesenteric lymph node, popliteal lymph node, sternum with bone marrow, femur with bone marrow, rib with cartilage, skeletal muscle, trachea, lung, heart, aorta, submandibular salivary gland (both), liver, gall bladder, pancreas, oesophagus, stomach, small intestine, large intestine, kidney (both), urinary bladder, prostate, testis (both), epididymis (both), vagina, uterus, ovary (both), pituitary gland, adrenal gland (both), thyroid with parathyroid gland, thymus, peripheral nerve, brain, spinal cord, eye with optic nerve (both), lacrimal gland (both), any tissue with gross lesions.
A complete necropsy with tissue preservation was performed on the dog which had to be sacrificed during the study.

HISTOPATHOLOGY: Yes
After the fixation, organ samples listed above (except femur with bone marrow) were taken, embedded in paraplast, sectioned at 3-5 microns, stained with haematoxylin and eosin, and subjected to a microscopical examination.

Other examinations:

In addition to the proposed tissue samples in the protocol, femur with bone marrow was preserved from all surviving animals at necropsy and histopathologically examined.

Statistics:

For each time point and parameter a trend test was conducted showing increasing or decreasing trends in location from control to the highest dose group. This test is sensitive to monotone dose-related treatment effects. Due to the small sample size (n=4) no other routine statistical tests were performed. For each group mean values and standard deviations were calculated for each parameter and time point.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Before sacrifice in moribund condition (day 31), the female of the high dose group (2500 ppm) showed apathy, lateral position, increased abdominal breathing, increased abdominal tension and tachycardia (180 beats/minute).
- Other clinical signs were observed only in the highest treatment group (2500 ppm). For males the following clinical signs were evident (described as incidences): vomiting (3 times, 2/4 animals, day 19, 20 and 21), yellowish discolouring of sclera and mucosa of the mouth (1 time, 1/4 animals, day 22 to 31).
- For females the following clinical signs were evident : yellowish discolouring of sclera and mucosa of the mouth (1 time, 1/4 animals, week 10 onwards), apathy (5 times, 2 animals, days 31 and 43 to 46), lateral and prone position (3 times, 2/ animals, days 31, 43, 44), tachycardia (1 time, moribund animal, day 31), abdominal breathing increased (1 time, moribund animal, day 31), abdominal tension increased (1 time, moribund animal, day 31).
- A slightly higher incidence of diarrhoea was observed for group 4 (2500 ppm) females at week 3 only. This finding is considered to be of no toxicological relevance.
- During acclimatisation, one male had a swelling on the left hindlimb. Another male had an injury on the tail and one female developed a conjunctivitis of both eyes. All lesions recovered either without treatment, upon local treatment with Degramycin ointment or upon i.m. injection of Ilcocillin.
- Slime in faeces was observed on single days in females of groups 2, 3, and 4 (100, 500 and 2500). However, in the absence of a dose-relationship this finding is considered incidental in nature.

Mortality:

mortality observed, treatment-related

Description (incidence):

One female dog in group 4 (2500 ppm) became moribund and was sacrificed on day 31 (week 5). All other animals survived to scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

For group 4 animals (2500 ppm), a loss in mean body weight was recorded for males from week 2 and for females from week 3 onwards. Total mean body weight loss was 7 and 16 % of pretest values in males and females, respectively.
At the end of treatment, a loss of body weight was noted in two males (34 and 16 %) and two females (34 and 7 %), compared with pretest values. After a transient loss in body weight at test week 3 to 5 (16 % of pretest weight) one male gained weight and, at the end of the treatment period, weighed more than at pretest. Weight gain for one female was slightly reduced and weight gains comparable to controls were recorded for one male and female (sacrifice in moribund condition).
Body weight development of other treated groups was similar to controls.
An overview of the final body weights can be found in Table 1 in 'any other information on results incl. tables'.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Compared to pretest values, mean food consumption of the high dose group (2500 ppm) was decreased by 11 to 59 % for males during treatment period and 4 to 51 % for females from week 2 onwards. A high individual variation in food intake was recorded among these animals, ranging from short periods to almost the entire treatment period and from slight to severe reduction. During week 7, one female refused medicated food intake. Therefore, on study days 45, 46, 48 and 49 this animal was force fed about 100 g control food, admixed with water.
No reduction of food intake was observed in the moribund female until day of moribund sacrifice.
Lower food consumption ratios for males and females of group 4 (2500 ppm) reflected the food and bodyweight changes recorded for dogs of this group.
No relevant changes in mean food consumption were recorded for 100 and 500 ppm.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The examination of cornea, lens, and fundus did not reveal any substance related findings.
No alterations of the pupillary reflex could be seen in control or treated dogs.
Detailed examination of the third eyelid under local anesthesia revealed conjunctivitis follicularis in all male and female dogs of the control and treated groups at pretest and at week 13. This finding is, therefore, not considered to be of experimental relevance.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Red blood cell parameters (erythrocyte count, haemoglobin, haematocrit) were decreased in males and females of the high dose group. A severe anaemia associated with macrocytosis, hyperchromasia of red blood cells was noted in two high dose females. One of these females showed an increased reticulocyte count, indicating compensatory erythropoietic activity.
Decreased numbers of blood platelets were noted in the high dose group with episodes of severe thrombocytopenia in individual dogs throughout the treatment period. Slightly lower blood platelet counts were also found in males receiving 500 ppm. Prothrombin time was prolonged in the high dose animals at weeks 4, 8 and 13. Also in the high dose, leukopenia with neutropenia and lymphocytosis occurred in one female at week 13, monocytosis and increased occurrence of young neutrophils in another female at weeks 8 and 13.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In blood chemistry, marked increase of plasma bilirubin, decreased plasma cholesterol and phospholipid levels, markedly higher activities of aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), alkaline phosphatase (AP) and gamma-glutamyl transpeptidase (GGT) were noted occasionally in two high dose males. Decreased activities of ASAT and/or ALAT were observed in the other 2 males of this group. The two anemic females (high dose) had markedly increased levels of plasma bilirubin. Increased levels of urea and AP activity as well as increased and/or decreased activities of ASAT and ALAT were noted among females of the high dose group. One high dose female had a very high creatine kinase activity at week 13. One female of the mid dose group (500 ppm) had increased activities of ALAT and AP at week 13. Plasma protein levels were elevated from week 8 in females at 500 ppm and 2500 ppm due to an increase of the globulin fraction.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Higher incidence of bilirubinuria, which correlated with the increased plasma bilirubin levels, was observed in the dogs of the high dose group.
No toxicological relevance was attributed to the increased incidence of ketones in urines of treated male groups because this finding was also observed in female controls.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Relative liver weight was increased for both sexes receiving 2500 ppm and a tendency to increased absolute and relative liver weight was recorded for both sexes receiving 500 ppm. Spleen weights were elevated at 2500 ppm. Reduced weights of testes were noted in males and of heart, thymus and thyroid in both sexes of the high dose group. Kidney weight of one high dose female was slightly increased. The affected organ weights are shown in Table 2 in 'any other information on results incl. tables'.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Body as a whole recorded as yellowish or whitish in 2/4 females of group 4 (2500 ppm). These findings are regarded to be signs of icterus or anaemia in congruence with microscopical findings in the liver, spleen and bone marrow listed in the section below and with laboratory data.
Liver recorded as mottled in (2/4) males and 1/4 females of group 4 (2500 ppm) corresponding to microscopical findings of the liver.
All other findings occurred in comparable numbers in all experimental groups and were similar to those occurring spontaneously in our colony of Beagle dogs. Thus, no experimental relevance is attributed to these findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopically, the following changes appeared to be treatment-related:
- Histopathological examinations revealed degenerative (hepatocellular necrosis, fibrosis) changes in 2/4 males and 2/4 females receiving 2500 ppm. In one female receiving 500 ppm recent necrosis of the liver was observed. Inflammatory changes of the liver, and proliferation of intrahepatic bile ducts were observed at 500 ppm and 2500 ppm.
- Inflammatory and/or degenerative changes in the skeletal muscle (myopathy), in the gastrointestinal wall (inflammation, oedema), in the thyroid, salivary gland and prostate (lymphohistiocytic infiltration) at 500 ppm and 2500 ppm. Additionally, in the high dose, similar changes occurred in the myocardium (inflammation), gall bladder of females (oedema), parathyroid gland of males (lymphohistiocytic infiltration) and autonomic ganglions in females (perivascular cell infiltration). These degenerative changes were mostly limited to the high dose group, except for one male in the control group which developed fibrosis in the salivary gland
- Dilatation of prostatic tubuli (reported as cystic dilatation of glandular tissue) and minimal tubular atrophy of the testis or reduced spermatogenesis at 500 ppm and 2500 ppm;
- Uterine atrophy at 2500 ppm.
- Atrophy of the thymic cortex at 500 ppm and 2500 ppm, atrophic lymphatic follicles and an increased occurrence of phagocytic cells in the mesenteric lymph node at 2500 ppm.
- Hypocellularity of the bone marrow in one high dose male and hypercellularity of the bone marrow in females of the mid and high dose groups.
- Splenic haemosiderosis in males and extramedullary haematopoiesis in females at 500 ppm and 2500 ppm.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

- Hypocellularity of the bone marrow in one high dose male and hypercellularity of the bone marrow in females of the mid and high dose groups.
3.12 mg/kg bw/day for males and 3.24 mg/kg bw/day for females, based on finding in the liver, thymus and spleen.

Details on results:

Additionally, a variety of other changes was found in this study. They commonly occur in our colony of Beagle dogs, and, neither their incidences nor their distribution and morphologic appearance gave any indication of a treatment-related association. These findings are not reported.

Key result

Dose descriptor:

NOAEL

Effect level:

100 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: equivalent to 3.12 mg/kg bw/day

Dose descriptor:

NOAEL

Effect level:

100 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: equivalent to 3.24 mg/kg bw/day

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 ppm

System:

hepatobiliary

Organ:

bile duct

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 ppm

System:

immune system

Organ:

thymus

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

2 500 ppm

System:

female reproductive system

Organ:

uterus

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Critical effects observed:

no

Lowest effective dose / conc.:

500 ppm

System:

male reproductive system

Organ:

other: prostate

Critical effects observed:

no

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

System:

haematopoietic

Organ:

bone marrow

Critical effects observed:

no

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

System:

male reproductive system

Organ:

testes

ANALYTICAL VERIFICATION OF DOSES:

- Concentration analysis results: The overall mean test substance concentrations were 102.2, 93.6 and 92.1% of nominal for 100, 500 and 2500 ppm.

- The homogeneity varied in the range from -4 % to +4 % of the mean concentrations.

- Stability results: the test substance was stable in rodent diet at room temperature over a period of 47 days.

Table 1. Final body weights at week 13

Feeding level [ppm]	0	100	500	2500
Male	12.85	12.08	12.40	9.80*
Female	11.53	11.68	11.78	8.00*

Wilcoxon: * p<0.05

 

Table 2. 90-day study in dogs - treatment-related changes in organ weights

	Males					Females
Parameter / Feeding level [ppm]	0	100	500	2500		0	100	500	2500
Heart [g]	113.8	105.4	111.5	82.39*		100.4	98.66	96.67	75.98*
Liver absolute [g]	341.0	348.4	398.2*	386.2		330.8	378.6	391.5*	369.5
Liver relative to body weight	28.53	30.26	33.90	40.01*		29.45	33.59	34.50	50.87*
Thymus [g]	11.26	10.11	8.58	5.28		11.16	12.94	10.73	3.21*
Spleen [g]	30.38	25.18	30.36	30.86		27.70	26.90	31.39	113.9
Thyroid [g]	1.202	0.910*	1.016	0.758*		0.979	1.096	1.154	0.700*
Testes [g]	20.83	18.43	20.28	14.28*

Wilcoxon test, * = p≤0.05

Conclusions:

In this GLP compliant OECD 409 study, the no-oberved-adverse-effect-level (NOAEL) was 100 ppm in both sexes, equivalent to dose levels of 3.12 mg/kg bw/day for males and 3.24 mg/kg bw/day for females, based on finding in the liver, thymus and spleen.

Executive summary:

In a GLP compliant study, performed according to OECD 409, groups of 4 male and 4 female Beagle dogs were treated orally for 90 days (13 weeks) with the test substance in the diet at concentrations of 0, 100, 500 and 2500 ppm (Altmann 1992). Clinical signs, body weight, food and water consumption were monitored throughout the study. Ophthalmoscopic examinations were performed before dosing commenced and towards the end of treatment. Haematology, clinical chemistry and urinalysis were performed throughout the study and at the end of the study. All animals were killed, subjected to necropsy and post mortem examination, major organs were weighed and a full range of tissues were examined microscopically. Also animals which died during the study were subjected to a full necropsy.

Mean achieved daily dose levels were 0, 3.12, 13.9 and 53.4 mg/kg bw/day (males) and 0, 3.24, 14.5 and 60.2 mg/kg bw/day (females), based on exposure to medicated feed containing 0, 100, 500 and 2500 ppm of the test substance. One female receiving 2500 ppm died during the study. Clinical signs were limited to animals receiving 2500 ppm and consisted of increased incidences in vomiting and a yellowish discolouring of sclera and mucosa of the mouth. The moribund female showed apathy, lateral and prone position, tachycardia and increased abdominal breathing and tension before the animal was sacrificed. Ophthalmoscopic examinations revealed no evidence of ocular toxicity. Body weights were only affected in animals receiving 2500 ppm, mainly because of a reduced food intake, which was decreased by 11 to 59 % for males and 4 to 51 % for females. A high variation in food intake was recorded among these animals. Red blood cell parameters (erythrocyte count, haemoglobin, haematocrit) were decreased in males and females of the high dose group. A severe anaemia associated with macrocytosis, hyperchromasia of red blood cells was noted in two females receiving 2500 ppm. Decreased numbers of blood platelets were noted in the 2500 ppm treatment group with episodes of severe thrombocytopenia in individual dogs throughout the treatment period. Prothrombin time was prolonged in the high dose animals at weeks 4, 8 and 13. Both sexes receiving 2500 ppm showed an increase of plasma bilirubin as well as increased and/or decreased activities of aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT). Plasma protein levels were elevated from week 8 in females at 500 ppm and 2500 ppm due to an increase of the globulin fraction. Higher incidence of bilirubinuria, which correlated with the increased plasma bilirubin levels, was observed in the dogs of the high dose group. Gross pathological investigations indicated mottled livers in 2/4 males and 1/4 females receiving 2500 ppm and indicated a yellowish or whitish colour of the body as a whole. Relative liver weight was increased for both sexes receiving 2500 ppm and a tendency to increased absolute and relative liver weight was recorded for both sexes receiving 500 ppm. Spleen weights were elevated at 2500 ppm for both sexes. Reduced weights of testes were noted in males and of heart, thymus and thyroid in both sexes of the high dose group. Histopathological examinations revealed degenerative (hepatocellular necrosis, fibrosis) changes in 2/4 males and 2/4 females receiving 2500 ppm. In one female receiving 500 ppm recent necrosis of the liver was observed. Inflammatory changes of the liver, and proliferation of intrahepatic bile ducts were observed at 500 ppm and 2500 ppm. Extramedullary haematopoiesis, haemosiderosis and cholestasis were observed among high dose animals. Inflammatory and/or degenerative changes in the skeletal muscle (myopathy), in the gastrointestinal wall (inflammation, oedema), in the thyroid, salivary gland and prostate (lymphohistiocytic infiltration) at 500 ppm and 2500 ppm. These degenerative changes were mostly limited to the high dose group, except for one male in the control group which developed fibrosis in the salivary gland. Additionally, in the high dose, similar changes occurred in the myocardium (inflammation), gall bladder of females (oedema), parathyroid gland of males (lymphohistiocytic infiltration) and autonomic ganglions in females (perivascular cell infiltration). Dilatation of prostatic tubuli (reported as cystic dilatation of glandular tissue) and minimal tubular atrophy of the testis or reduced spermatogenesis at 500 ppm and 2500 ppm. Uterine atrophy at 2500 ppm. Atrophy of the thymic cortex at 500 ppm and 2500 ppm, atrophic lymphatic follicles and an increased occurrence of phagocytic cells in the mesenteric lymph node at 2500 ppm. Hypocellularity of the bone marrow in one high dose male and hypercellularity of the bone marrow in females of the mid and high dose groups. Splenic haemosiderosis in males and extramedullary haematopoiesis in females at 500 ppm and 2500 ppm. The no-observed-adverse-effect-level (NOAEL) was 100 ppm in both sexes, equivalent to dose levels of 3.12 mg/kg bw/day (males) and 3.24 mg/kg bw/day (females), based on findings in the liver, thymus and spleen.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

3.12 mg/kg bw/day

Study duration:

subchronic

Species:

dog

Quality of whole database:

GLP compliant OECD 409 study

System:

other: hepatobiliary, immune, haematopoietic system

Organ:

liver

spleen

thymus

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

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

All available data was assessed and the studies representing the worst-case effects were included as key studies. The other studies are included as supporting information. The key studies are considered to be worst-case and were selected for the CSA

 

Repeated dose toxicity: oral

3-month feeding in dogs

In a GLP compliant study, performed according to OECD 409, groups of 4 male and 4 female Beagle dogs were treated orally for 90 days (13 weeks) with the test substance in the diet at concentrations of 0, 100, 500 and 2500 ppm (Altmann 1992). Clinical signs, body weight, food and water consumption were monitored throughout the study. Ophthalmoscopic examinations were performed before dosing commenced and towards the end of treatment. Haematology, clinical chemistry and urinalysis were performed throughout the study and at the end of the study. All animals were killed, subjected to necropsy and post mortem examination, major organs were weighed and a full range of tissues were examined microscopically. Also animals which died during the study were subjected to a full necropsy.

Mean achieved daily dose levels were 0, 3.12, 13.9 and 53.4 mg/kg bw/day (males) and 0, 3.24, 14.5 and 60.2 mg/kg bw/day (females), based on exposure to medicated feed containing 0, 100, 500 and 2500 ppm of the test substance. One female receiving 2500 ppm died during the study. Clinical signs were limited to animals receiving 2500 ppm and consisted of increased incidences in vomiting and a yellowish discolouring of sclera and mucosa of the mouth. The moribund female showed apathy, lateral and prone position, tachycardia and increased abdominal breathing and tension before the animal was sacrificed. Ophthalmoscopic examinations revealed no evidence of ocular toxicity. Body weights were only affected in animals receiving 2500 ppm, mainly because of a reduced food intake, which was decreased by 11 to 59 % for males and 4 to 51 % for females. A high variation in food intake was recorded among these animals. Red blood cell parameters (erythrocyte count, haemoglobin, haematocrit) were decreased in males and females of the high dose group. A severe anaemia associated with macrocytosis, hyperchromasia of red blood cells was noted in two females receiving 2500 ppm. Decreased numbers of blood platelets were noted in the 2500 ppm treatment group with episodes of severe thrombocytopenia in individual dogs throughout the treatment period. Prothrombin time was prolonged in the high dose animals at weeks 4, 8 and 13. Both sexes receiving 2500 ppm showed an increase of plasma bilirubin as well as increased and/or decreased activities of aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT). Plasma protein levels were elevated from week 8 in females at 500 ppm and 2500 ppm due to an increase of the globulin fraction. Higher incidence of bilirubinuria, which correlated with the increased plasma bilirubin levels, was observed in the dogs of the high dose group. Gross pathological investigations indicated mottled livers in 2/4 males and 1/4 females receiving 2500 ppm and indicated a yellowish or whitish colour of the body as a whole. Relative liver weight was increased for both sexes receiving 2500 ppm and a tendency to increased absolute and relative liver weight was recorded for both sexes receiving 500 ppm. Spleen weights were elevated at 2500 ppm for both sexes. Reduced weights of testes were noted in males and of heart, thymus and thyroid in both sexes of the high dose group. Histopathological examinations revealed degenerative (hepatocellular necrosis, fibrosis) changes in 2/4 males and 2/4 females receiving 2500 ppm. In one female receiving 500 ppm recent necrosis of the liver was observed. Inflammatory changes of the liver, and proliferation of intrahepatic bile ducts were observed at 500 ppm and 2500 ppm. Extramedullary haematopoiesis, haemosiderosis and cholestasis were observed among high dose animals. Inflammatory and/or degenerative changes in the skeletal muscle (myopathy), in the gastrointestinal wall (inflammation, oedema), in the thyroid, salivary gland and prostate (lymphohistiocytic infiltration) at 500 ppm and 2500 ppm. These degenerative changes were mostly limited to the high dose group, except for one male in the control group which developed fibrosis in the salivary gland. Additionally, in the high dose, similar changes occurred in the myocardium (inflammation), gall bladder of females (oedema), parathyroid gland of males (lymphohistiocytic infiltration) and autonomic ganglions in females (perivascular cell infiltration). Dilatation of prostatic tubuli (reported as cystic dilatation of glandular tissue) and minimal tubular atrophy of the testis or reduced spermatogenesis at 500 ppm and 2500 ppm. Uterine atrophy at 2500 ppm. Atrophy of the thymic cortex at 500 ppm and 2500 ppm, atrophic lymphatic follicles and an increased occurrence of phagocytic cells in the mesenteric lymph node at 2500 ppm. Hypocellularity of the bone marrow in one high dose male and hypercellularity of the bone marrow in females of the mid and high dose groups. Splenic haemosiderosis in males and extramedullary haematopoiesis in females at 500 ppm and 2500 ppm. The no-observed-adverse-effect-level (NOAEL) was 100 ppm in both sexes, equivalent to dose levels of 3.12 mg/kg bw/day (males) and 3.24 mg/kg bw/day (females), based on findings in the liver, thymus and spleen.

 

1-year feeding in dogs

In this GLP-compliant OECD 452 study, the test substance was administered orally by admixture in the diet to Beagle dogs at concentrations of 0, 20, 200 and 1000 and ppm (Altmann 1994). Each dose group consisted of 4 animals of each sex. The control group and the highest dose group included 2 additional dogs for evaluation of reversibility of adverse effects, with a recovery period of 28 days. Mortality, clinical signs and food consumption were recorded daily, individual body weights recorded weekly. Haematological, clinical chemistry, urinalysis and electrocardiography investigations were performed on all animals in weeks 13, 26, 52 and 56 (only for the additional animals in the control group and the highest dose group). Ophthalmoscopy examination were performed towards the end of the treatment period ant at the end of the follow-up periods in the animals of the recovery group. All animals were subjected to necropsy and post mortem examination, major organs were weighed and selected tissues examined histopathologically.

Mean achieved daily dose levels (corrected for the analytical purity of the test substance) were 0, 0.57, 5.33 and 27.9 mg/kg bw/day in males and 0, 0.57, 5.03 and 27.4 mg/kg bw/day in females at dose levels of 0, 20, 200 and 1000, respectively. One male in the highest treatment group died on day 229 (wk 33) because of acute bronchopneumonia which was not treatment-related. No ambiguous effects were observed in body weight gain. Mean body weight gains in females were higher than in the controls, but lacked a dose-relationship. Body weight losses, most often because of refused food intake, were regained to the previously recorded body weights. There were some incidences in the highest dose group with slight changes in haematological parameters which were not considered to be treatment related. One female suffered from severe anaemia also showed marked changes in blood chemistry parameters: increased plasma bilirubin levels (total and direct), increased globulin and total protein levels, decreased albumin levels, increased activities of alanine aminotransferase and alkaline phosphatase, and slightly lower T3 and T4 levels. All these alterations were reversible during the following 6 months of treatment. The male which died during the treatment period had a markedly increased activity of creatine kinase at week 13, and skeletal muscle myopathy was found at histopathological examination. There were no effects on any urine parameters, bone marrow analysis or electrocardiography. No effects of toxicological relevance were evident among the organs weighed, since all minor effects were within the expected biological range. At necropsy, scarring of the liver of one male in the highest treatment group was observed, probably caused by heavily infiltration by inflammatory cells. There were no other gross findings which could be considered to represent a treatment-related effect.

Treatment-related histopathological findings were restricted to animals of group 4 (1000 ppm) and consisted of a myopathy (2/6 males), an increased severity of inflammatory cell infiltration of the liver (3/6 males), a hyperplasia of intrahepatic bile ducts (1/6 males), and an increased splenic and hepatic haemosiderosis (1/6 males, 1/4 females). In the dog which died prematurely during the treatment period, a marked acute bronchopneumonia was the obvious cause of death. It was associated with acute reactive hyperplasia of the cervical and mesenteric lymph node. All other microscopical findings observed in males and females in this study were considered as toxicologically irrelevant.

The test substance at 1000 ppm in the diet provides daily dose levels that exceed the MTD, and the results are a suitable basis on which to establish dose levels for a 1-year study. The no-observed-adverse-effect-level (NOAEL) was 200 ppm in both sexes, equivalent to dose levels of 5.33 mg/kg bw/day (males) and 5.03 mg/kg bw/day (females), based on the findings of blood chemistry, myopathy and effects in the hepatobiliary system.

 

28 -day gavage in rats

In this GLP compliant OECD 407 study, the test substance was administrated daily by oral gavage to rats (Tif: RAif (SPF)) at concentration of 0 (control), 10, 100 and 600 mg/kg bw/day for 28 consecutive days (Fankhauser 1992). Destilled water containing 0.5% carboxymethylcellulose and 0.1% Tween 80 was used as vehicle for preparation of the suspensions. Each group consisted of 10 animals per sex. Mortality, the state of health or behaviour, or any other reaction to the treatment was monitored daily. Body weight, food and water consumption were recorded weekly (midweek). Ophthalmoscopy examinations were done on Day -6 and Day 24. Haematological, clinical chemistry and urinalysis investigations were performed on all animals towards the end of the treatment period (Day 24). All animals were subjected to necropsy and post mortem examination, major organs were weighed and selected tissues examined histopathologically. Mean achieved daily dose levels (corrected for the analytical purity of the test substance) were 0, 98.3%, 98.4% and 98.3 at dose levels of 10, 100 and 600 mg/kg bw/day respectively.

One non-treatment related death occurred in the highest treatment groups due to improper intubation. A total of 7/10 males and 5/10 females of group 4 (600 mg/kg) showed a reddening of the ears. Mean body weights of 7% and 10% below the control values were recorded at week 4 for males and females of group 4. Lower food intakes were recorded for males of group 4 (600 mg/kg) during the first two weeks of the study. Subsequent intakes were similar to control values. For females of group 4 (600 mg/kg), lower food intakes were recorded for the duration of the study, resulting in an overall intake of 20% below the control value. Males and females of group 4 (600 mg/kg) had a mild anaemia with a tendency to macrocytosis and hyperchromasia of red blood cells. The anaemia was associated with higher values for reticulocyte counts, indicating a compensatory erythropoietic activity. Furthermore, slightly higher values for white blood cell counts were recorded for both sexes of the high dose group (600 mg/kg). Higher plasma levels for bilirubin, protein, albumin and cholesterol, and lower plasma levels for chloride were recorded for males and females of group 4 (600 mg/kg). Furthermore, minimally lower plasma glucose levels and increased activities of alkaline phosphatase were noted in males of the high dose group (600 mg/kg), and minimally lower plasma potassium levels were recorded for females of this group. Urinalysis revealed presence of bilirubin in two females, and a slight increase of urine density in males at 600 mg/kg bw.

Organ weights and gross pathological findings were mainly confined to group 4 (600%). The livers in the group 4 were enlarged in 7/10 males and 7/10 females and mottled in 2/10 males, the organ weights were increased with 40 to 59%, for males 49% to 72% for females. In group 3 (100 mg/kg) the only the organ weight were increased by 12% and 10% for males of group 3. All other effects in organ weights were limited to group 4. Small thymus were seen in 3/10 males and 2/10 females with a relative decrease in organ weight of 43 and 36% respectively. Mottled lungs observed in 5/10 males and 2/10 females. Carcass weights (-11.5 to -12.5%), kidney (23% males, 12% females) and spleen (23 to 39% males, 22 to 42% females). All other findings occurred in comparable numbers in all experimental groups and were similar to those occurring spontaneously in our colony of rats. Thus no experimental relevance is attributed to these findings.

Histopathological findings revealed hypertrophy of hepatocytes of the centrilobular region of the liver was seen in 5/10 males and 3/10 females of group 3 (100 mg/kg) and all males and females of group 4 (600 mg/kg). The lesion was moderate in 3/10 males and 7/10 females of group 4 and slight in all other affected animals. Thymic atrophy was observed in 2/10 males of group 3 (100 mg/kg) and 5/10 males and 4/10 females of group 4 (600 mg/kg). This change was found to be marked in one female and moderate in one animal of either sex in group 4 and slight in the other ones. Slight hyperplasia of the splenic white pulp was found in 4/10 males and 3/10 females of group 3 (100 mg/kg) and 8/10 males and 4/10 females of group 4 (600 mg/kg). The testis showed slightly reduced spermatogenesis in 9/10 males of group 4 (600 mg/kg). Seven out of these 9 animals presented additionally with reduction of spermatozoa in the epididymis. A slight decrease of nephrocalcinosis normally found in almost all of our female rats was observed in female group 4 (600 mg/kg) when compared with the control and the other treated groups. This decrease was considered to be of no toxicological relevance. No other treatment-related microscopic lesions occurred.

The level of 10 mg/kg bw/day was the no observable effect level (NOAEL), based on changes in liver, thymus and spleen at 100 mg/kg bw/day.

 

Additional information on repeated dose toxicity obtained from carcinogenicity studies in section 7.7

2 -year carcinogenicity – rats

This GPL compliant OECD 453 study was conducted to assess the carcinogenicity and chronic toxicity of the test substance when administered in the diet to rats (strain: Tif: RAIf (SPF), at dietary concentrations of 0, 10, 100, 1000 and 3000 ppm (= mg/kg food) for 24 months (for females equivalent to 0.357, 3.73, 39.3 and 128 mg/kg body weight/day; for males equivalent to 0.430, 4.45, 47.1 and 154 mg/kg body weight/day) (Gerspach 1995).

Dietary levels of 1000 and 3000 ppm of the test substance caused inappetence with the resulting reduction of body weight gain, especially marked in males and females in the highest treatment group. Changes to the haematology and blood chemistry profiles may have been a reflection of the nutritional status of the animals these groups. Higher liver, kidney and spleen to body weight ratios were noted in all 3000 ppm and for males of 1000 ppm exposed groups. The marginal increased incidences of neoplasms in the adrenal medulla, cerebral meninges and lymphoreticular tissue were considered of no toxicological relevance, especially with regard to the historical control data in the conducting laboratory. Microscopical examination revealed the liver to be the target organ as indicated by a significantly increased incidence of hepatocellular hypertrophy in both sexes at 1000 and 3000 ppm, an increased incidence of foci of cellular change in both sexes at 3000 ppm, and increased incidences of benign hepatomas and biliary cysts in females at 3000 ppm. Mainly based on liver toxicity evidenced by hypertrophy of hepatocytes in both sexes at 1000 ppm, the No Observed Adverse Effect Level (NOAEL) is 100 ppm, equivalent to 3.7 or 4.45 mg/kg bw/day for males and females, respectively.

 

18 months carcinogenicity - mice

In this GLP compliant, OECD 451 study, groups of 60 male and 60 female mice (strain Tif:MAGf) were administered with the test substance orally for 18 months, by admixture in the diet, at concentrations of 0, 10, 100, 2000 or 5000 ppm (dietary equivalent of 1.24, 12.0, 254 and 678 mg/kg bw/day for males and 1.17, 11.4, 243 and 673 mg/kg bw/day for females) (Gerspach 1995).

The treatment did not adversely affect survival or appearance or behaviour of the animals. Compared with the controls, a significantly higher number of group 5 males (5000 ppm) survived the treatment period. At the end of the study, mean body weight were decreased relatively to the control for animals in the groups receiving 2000 and 5000 ppm (group 4 and 5), for males -7% and -23% and for females -15% and -44%, respectively. Food consumption was not affected by treatment at any dose level except in the high dose (5000 ppm) females. For the majority of the treatment period, higher food consumption ratios were obtained for males and females of group 5 (5000 ppm). The treatment did not influence the haematological profile of the mice. Significantly higher organ weights and organ to body weight ratios were obtained for liver of males and females of groups 4 and 5 (2000 and 5000 ppm) and for adrenals of males of groups 4 and 5. Significantly higher ratios were also obtained for spleen of males of group 5 (5000 ppm) and for kidneys of females of group 5. Necropsy revealed a higher incidence of liver masses in male groups 4 and 5 (2000 and 5000 ppm) and female group 5. An increased incidence of animals showed enlarged livers in groups 4 and 5 and mottled livers in group 5. Splenic enlargement was noted in male groups 4 and 5. Microscopical examination revealed a significantly increased incidence and severity of liver cell hypertrophy in male and female groups 4 and 5. Furthermore, an increased number of liver nodules in animals of group 5 was observed. In addition, higher incidences were recorded for chronic inflammation of the glandular stomach and hyperplasia of the gastric mucosa in male group 5, extramedullary haematopoiesis in the spleen in male and female groups 4 and 5, hypercellularity of the bone marrow in male groups 4 and 5 and female group 5, and haemosiderosis of the spleen in male group 5 and female groups 4 and 5. A significantly increased incidence of benign and malignant liver tumours in both male and female groups 5 and of malignant tumours also in male group 4 were observed.

Mainly based on toxicity to the liver evidence by increased tumour formation in males, the No Observed Adverse Effect Level (NOAEL) is 100 ppm, equivalent to a lifetime daily dose of 11.4 mg/kg bw/day.

 

Repeated dose toxicity: dermal

28 -day dermal study in rats

In the present study albino rats (Tif: RAif (SPF)) were dermally exposed to 0, 10, 100 and 1000 mg/kg bw per exposure to the test compound (Hagemann 1993). The control group was exposed to vehicle (distilled water) only. Each group consisted of 5 animals per sex. The test article/vehicle suspension was applied under occlusive dressing to the shaved back skin for a period of 4 weeks on a 5 day /week basis. The exposure period was 6 hours per day. The dressings were removed after 6 hours and the application areas were cleaned with lukewarm water. Control analysis of the test article concentration in the vehicle was carried out at all dose levels. The animals were checked for clinical signs and mortality on daily basis. Approximately 17 hours after removing the gauze patches, local skin reactions at the application site of the test article were evaluated according to Draize method. Body weights, food consumption were recorded weekly. Laboratory investigations (haematology, blood chemistry) were carried out on all surviving animals of each dose group at the end of the treatment period. At the end of the test period the surviving controls and treated animals were bled under ether anaesthesia and subjected to detailed autopsy. Organ samples were prepared and subjected to a microscopical examination.

The content of active ingredient in all samples was found to be in agreement with the nominal concentration. The test article was stable in the vehicle (distilled water) for 5 days at room temperature. During the whole observation period no treatment-related symptoms were observed. Furthermore, signs of local irritation were transient and a dose-response relationship was missing. One female in the 10 mg/kg bw group died, no treatment-related mortality occurred during this study. Body weight, food consumption, haematological profile, and blood chemistry parameters investigated were not influenced by the treatment. Organ weights were not affected by the treatment. All macroscopical and microscopical pathology findings were considered to be incidental in nature. In particular, no evidence of dermal toxicity was observed.

It is concluded that the "no observed effect level" (NOEL) for the test substance in this study is higher than the limit dose of 1000 mg/kg bw

Justification for classification or non-classification

There were clear effects in the 90 day dog study at 2500 ppm (53/60 mg/kg bw/day), including severe anaemia and termination of one animal; however this dose level is above the allometrically adjusted classification threshold for the dog (2.9-29 mg/kg bw/day), and hence does not trigger classification. The effects at the lower dose in the dog study (500ppm = 13.9/14.3 mg/kg bw/day) were degenerative and/or inflammatory changes of the liver and proliferation of intrahepatic bile ducts, atrophy of the thymic cortex, and splenic haemosiderosis; the range & severity of effects was less severe than seen at 2500 ppm, and considered to be below the severity level required for STOT classification.

Based on the available information classification upon repeated exposure is not warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.