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

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

6-methyl-4-[(E)-[(pyridin-3-yl)methylidene]amino]-2,3,4,5-tetrahydro-1,2,4-triazin-3-one

EC Number:

602-927-1

Cas Number:

123312-89-0

Molecular formula:

C10H11N5O

IUPAC Name:

6-methyl-4-[(E)-[(pyridin-3-yl)methylidene]amino]-2,3,4,5-tetrahydro-1,2,4-triazin-3-one

Test material form:

solid: particulate/powder

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

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.

Doses / concentrations 2

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.

Doses / concentrations 3

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.

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

1

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

2

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

Target system / organ toxicity

1

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

2

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

3

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

4

Critical effects observed:

no

Lowest effective dose / conc.:

500 ppm

System:

male reproductive system

Organ:

other: prostate

5

Critical effects observed:

no

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

System:

haematopoietic

Organ:

bone marrow

6

Critical effects observed:

no

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

System:

male reproductive system

Organ:

testes

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.