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Toxicological information

Carcinogenicity

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Description of key information

The available information regarding genotoxicity does not gives rise to carcinogenic potential. It cannot be excluded that chronic administration might deteriorate the inflammatory processes seen in repeat-dose studies leading to hyperplasia/neoplastic lesions. However, in a 2-year feeding study with a structurally related substance showing the same inflammatory effects after repeated exposure, no indication of carcinogenesis was evident.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1987 - 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 451 (Carcinogenicity Studies)
GLP compliance:
yes
Species:
rat
Strain:
other: Tif: RAIf (SPF), hybrids of RII/1 x RII/2
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Animal Production, CIBA-GEIGY Limited, 4332 Stein / Switzerland
- Age at study initiation: approx. 4-5 weeks
- Weight at study initiation: 88.4 - 125.8 g in males; 76.1 - 114.5 g in females
- Housing: groups of 5 animals
- Diet (e.g. ad libitum): Pelleted, certified standard diet (Nafag No. 890 Tox) was provided ad libitum.
- Water (e.g. ad libitum): Tap water was given ad libitum
- Acclimation period: ten days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 10
- Air changes (per hr): 16 - 20
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Fresh diets were prepared for week 1 and then at biweekly intervals until week 11. Diets were then prepared every 3 weeks until week 17 then subsequent mixes were prepared at monthly intervals.
- Mixing appropriate amounts with (Type of food): The pulverised food was homogeneously mixed with the appropriate concentrations of the test article and about 25 % water was added before pelleting to ensure the necessary pellet quality.
- Storage temperature of food: The air-dried food was stored in stainless steel containers at room temperature until used.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Analyses of the diet concentration were undertaken periodically with the diet batches applied during the study. These analyses were carried out in the laboratories of Analytical Services Rosental , CIBA-GEIGY Limited, Basle/ Switzerland. Analytical method: KS-152/1 (via LC).
- Average achieved intakes were: 4.77, 28.4 and 192 mg/kg bodyweight per day for males and 5.05, 29.2 and 200 mg/kg bodyweight per day for females, of groups 5, 30 and 200 mg/kg bw, respectively.
Duration of treatment / exposure:
24 months (105 to 106 weeks)
Frequency of treatment:
daily with diet
Dose / conc.:
5 mg/kg bw/day (nominal)
Remarks:
Average Dose level accheived: 4.77 mg/kg bw/day for males and 5.05 mg/kg bw/ day for females
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Average Dose level accheived: 28.4 mg/kg bw/day for males and 29.2 mg/kg bw/ day for females
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Average Dose level accheived: 192 mg/kg bw/day for males and 200 mg/kg bw/ day for females
No. of animals per sex per dose:
50
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Dose levels were based on the results of the following previously conducted studies:
1) Project no. 801458 Acute, oral toxicity study in rats: LD50 above 7000 mg/kg bw.
2) Project no. 801624 Acute, oral toxicity study in rats: LD50 above 5000 mg/kg bw.
3) Project no. 801625 28-day range finding study in rat. Test substance was administered at dosage levels of 1000, 3000 and 10000 ppm, corresponding to 80-83, 206-211 and 788-838 mg/kg bw.
4) Project No. B78/0848 Centraal Instituut voor Voedingsonderzoek, Zeist, February 1980. Subchronic (13 week) oral toxicity study in rats
5) Project No. B80-1927 Centraal Instituut voor Voedingsonderzoek, Zeist, June 1982. Restricted subchronic (13 week) oral toxicity study in rats.
6) Project no. 840701 3-month toxicity study in rats (Draft Report).
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly (midweek) for the first 3 months and monthly thereafter.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly for the first 3 months and monthly thereafter.

FOOD CONSUMPTION RATIOS: Yes, mean of individual calculations according to the following formula: weekly food consumption (g) x 1000 / (midweek bodyweight (g) x 7); Unit: g food/kg bodyweight/day

MICROBIOLOGICAL INVESTIGATIONS
After 60 weeks of treatment it was decided to sacrifice animals of group 4 (200 mg/kg) exhibiting signs of hunched posture and distended abdomen. In order to exclude a disease related reason for morbidity, these animals were subjected to sterile dissection for microbiological investigations and blood samples were withdrawn for serological determination of viral antibodies.
Serotiters were determined for the following infective agents:
RCV / SDA: Coronavirus / Sialodacryoadenitis
MHV: Hepatitis
PVM: Pneumonia
Sendai: Parainfluenza
LCM: Lymphocytic choriomeningitis
Tyzzer's: Bacillus piliformis
KRV / H-1: Kilham / Toolan viruses

WATER CONSUMPTION: Yes
- Time schedule for examinations: monthly

TEST ARTICLE INTAKE: calculated according to the following formula: nominal dose (ppm) x food ccnsumption (g/animal week) / (bodyweight (g) x 7); Unit: mg test article/kg bodyweight/day

HAEMATOLOGY: Yes, on the surviving animals
- Time schedule for collection of blood: in the morning at week 27, week 52, week 78, week 83 and week 104
- Anaesthetic used for blood collection: Yes, ether
- Parameters checked: Erythrocyte Count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Reticulocytes, Leucocyte Count, Differential Leucocyte Count, Thrombocyte Count
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
At the end of the test period the surviving controls and treated animals were bled under ether anesthesia, weighed and subjected to a detailed autopsy. Unless prevented by advanced autolysis, cannibalism or for technical reasons a complete autopsy with tissue preservation was performed also on all animals which died during the test period or which had to be sacrificed in moribund condition.

HISTOPATHOLOGY: Yes
- Organs and tissues checked: skin, mammary area, spleen, mesenteric lymph node, axillary lymph node, popliteal lymph node, sternum with bone marrow, femur with joint, skeletal muscle, trachea, lung, heart, aorta, submandibular salivary gland (both), liver, pancreas, oesophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (caecum, colon, rectum), kidney (both), urinary bladder, prostate, seminal vesicle, testis (both), epididymis (both), vagina, uterus (corpus and cervix), ovary, both (with fallopian tubes), pituitary gland, adrenal gland (both), thyroid with parathyroid gland, thymus, peripheral nerve, brain, spinal cord (2 levels), eye with optic nerve, both (and contiguous Harderian gland), orbital gland (both), extraorbital lacrimal gland (both), Zymbal gland (both), muzzle (with tongue), organs and tissues with macroscopical changes
Statistics:
For each time point and parameter an univariate statistical analysis is performed. Nonparametric methods are applied, to allow for non normal as well as normal data distribution.
Each treated group is compared to the control group by Lepage's two-sample test (Y. Lepage, Biometrika (1971) 58: pp. 213-217). This test is a combination of Wilcoxon and Ansari-Bradley statistics, i.e. a combined test for location and dispersion.
Increasing or decreasing trends in location from control to the highest dose group are tested by Jonckheere' s test (A. R. Jonckheere, Biometrika (1954) 41: pp. 133-145) for ordered alternatives. This test is sensitive to monotone dose-related treatment effects.
Survival analysis is performed by the regression model (partial likelihood) introduced by Cox (D.R. Cox, Journal of the Royal Statistical Society Series B (1972) 34: pp. 187-220) in order to compare survival time of treated animals with control animals.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
During the second year of the study a number of males and females from group 200 mg/kg displayed a distended abdomen, piloerection, discoloured eyes in females (reported as "pale"), hunched back and bodyweight loss. These signs generally occurred in the few weeks preceding death. In addition, predominantly in males of groups 30 and 200 mg/kg an increased incidence of swollen hindlimbs was recorded. This was mainly an unilateral finding and for the majority of affected rats was a transient phenomenon. The lower incidence of palpable masses recorded for males and females of group 4 (200 mg/kg) was related to the early mortality occurring in this group, and hence, the reduced "time at risk " of developing tumours. Among males of group 4 (200 mg/kg) a higher incidence of unilateral eye lesions ocurred, the majority of which resulted from mechanical damage during the withdrawal of blood samples from the orbital sinus. Consequently, ths finding was not considered to be related to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Following completion of ten months of treatment, males and females of group 4 (200 mg/kg) began to die. By week 82 all animals of this group were dead or sacrificed in a moribund condition. The survival of males of groups 2 and 3 (5 and 30 mg/kg) was similar to that of the control group with 52% and 54% respectively surviving to terminal necropsy compared with 48% of the male controls.
Survival of group 2 and 3 females (5 and 30 mg/kg) was 44 and 42% respectively compared with the survival of 66% in the female control group. However, a number of group 2 females were sacrificed for humane reasons in moribund condition (according to internal standard operating procedures) because of tumors of the mammary gland. The relatively high number of moribund females contributing to the reduced survival of group 2 females was not considered to be related to the treatment for the following reasons:
- the statistical analysis of tumors of the mammary gland indicated no relationship to treatment
- other histopathological findings failed to indicate a common cause of death
- the sacrifice in moribund condition biased the normal mortality
Likewise no biological significance was attributed to the survival analysis of group 3 females, that revealed a statistical significance for increased mortality when compared with the control group. The plot on female mortality reveals a number of obviously premature deaths in groups 2 and 3. However, analysis of these early fatalities (5 and 6 females in groups 2 and 3, respectively which died before week 70) revealed ante mortem / histopathological findings which were all considered not treatment-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
From week 6, lower bodyweight gain for males of group 4 (200 mg/kg) resulted in mean bodyweights significantly lower than controls from week 16 onwards. During the remaining treatment period mean bodyweights of this group remained static. For the first 6 weeks of treatment positive trends as well as significantly increased mean bodyweights for group 4 females (200 mg/kg) were recorded. These findings are considered incidental since an uneven distribution of mean bodyweights was recorded already for the pretreatment week (week -1). Thereafter, lower bodyweight gains were recorded resulting in significantly lower mean values from week 40 onwards.
Mean bodyweights for males and females of groups 2 and 3 (5 and 3 0 mg/kg) were not influenced by treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
During the period of weeks 1 to 80, males of group 4 (200 mg/kg) consumed progressively less food than the controls with an overall intake 11 % lower than that of the control group. Females of group 4 (200 mg/kg) however, had food intakes essentially similar to control values apart from a period between weeks 33 and 64 when slightly lower intakes were recorded. The overall intake (weeks 1 - 80) for this group was onl y 4 % lower than the control value. Food intake values for males and females of groups 5 and 30 mg/kg were similar to those of the control group.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Bodyweight gain was lower than could be expected from lowered diet intake in males and females of group 4 (200 mg/kg). This resulted in higher food consumption ratios during the latter part of this groups survival. Food consumption ratios for other treated groups were similar to those of the respective control groups.
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
The water intakes for treated males were similar to those of the control group with overall intakes differing from the control value by less than 3%. Among treated females, animals of group 2 (5 mg/kg) showed a tendency to consume slightly more water than the controls (overall intake 8.9% higher than the control value). The water intakes for females of groups 3 and 4 (30 and 200 mg/kg) were similar to those of the control group. Therefore, the difference recorded for group 2 was considered of no toxicological relevance.
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Among males and females of group 4 (200 mg/kg) treatment produced a slight anemia with hypochromasia and microcytosis associated with higher incidences of anisocytosis, poikilocytosis, and polychromasia of the red blood cells. In addition, a marked leucocytosis occurred with neutrophilia and relative lymphopenia. Hypersegmentation of neutrophils was also noted in most animals. Furthermore, an increase in the number of thrombocytes was recorded for animals of this dose group. To some extent these effects were also noted in animals of group 3 (30 mg/kg).
Differences occurring in other treated groups which attained a level of statistical significance from control values were attributed to spontaneous variation and were considered to be of no clinical relevance.
The lymphatic leukemia detected at terminal laboratory investigation at week 104 in one female of group 3 (30 mg/kg) was not considered treatment-related.
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The major findings were described as masses, nodules or enlargement of various organs in a number of rats from group 4 (200 mg/kg), primarily of the mesenteric lymph node, which was also affected in some animals from group 3 (30 mg/kg). The other affected tissues included liver, spleen, thymus, salivary gland, heart, adrenal gland, axillary lymph node, kidney and caecum. Some of these organs also showed mottled appearance, fibrinous adhesions and scars. A number of male and female rats from group 4 (200 mg/kg) presented at autopsy with pleural effusion and ascites, and with oedematous or mottled lungs. Male rats from this dose group showed increased incidence of small seminal vesicles, as well as dilatation of the small intestine. Some male (9/50) and female (2/50) rats from group 3 (30 mg/kg) which survived the whole test period revealed thickening or hardening of one or more extremities and/or of the tail (ankylosing osteoarthritis). Except for one control male with a thickened hindlimb no such changes were seen in other experimental groups.
All other findings were present in comparable numbers in all experimental groups and were similar to those occurring spontaneously in our colony of rat. Thus no toxicological relevance was attributed to these findings.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In animals from groups 3 and 4 (30 and 200 mg/kg) a number of non-neoplastic findings was recorded which consisted of recurrent or chronic inflammatory lesions in various organs. They included circumscribed granulomatous lesions with coagulative necrosis, accompanied by degenerating polymorphs and surrounded by macrophages. The appearance of these cells varied from phagocytes with finely eosinophilic cytoplasm to epitheloid cells or foam cells. Eosinophils were generally scanty and giant cells were rare. Special stains did not give evidence of the presence of acid-fast bacilli or other microorganisms. The size of the lesions ranged from small "initial" granulomas to advanced lesions with necroses extending into the adjacent tissue. A coincidence of small and large lesions at the same site was not uncommon.
The necrotic areas themselves presented frequently as accumulations of densely packed basophilic material suggestive of nuclear debris. The type of lesion described above is termed "chronic necrotizing inflammation" in this report. In some tissues infarct-like necrosis occurred with a lesser degree of cellular reaction. These findings are reported as "necrosis". Chronic necrotizing inflammation was prominent in both sexes from group 4 (200 mg/kg). It occurred most extensively in the mesenteric lymph nodes, affecting all male (47/47) and female (50/50) rats from group 4 (200 mg/kg) and 30/50 males and 18/48 females from group 3 (30 mg/kg). Various other organs were affected by chronic necrotizing inflammation in group 4 (200 mg/kg) rats. The second most frequently involved site in both sexes was the liver, followed by the ovaries in females. Furthermore, frequently involved sites in both sexes were the salivary gland, adrenal gland (incidence ratio between males and females approximately 1:2), spleen, other lymph nodes, teeth (pulp), myocardium, nasopharynx and kidneys. At a low incidence, inflammatory foci were observed at vastly different sites such as mediastinum, peritoneum, pancreas, choroid plexus and brain, adenohypophysis, thyroid gland, skeletal muscle, and vagina. In group 3 (30 mg/kg) chronic necrotizing inflammation was restricted to the mesenteric lymph nodes except for one female rat with additional chronic necrotizing inflammation of the submandibular salivary gland.
Granulomatous reaction associated with the presence of foam cells at the periphery of necrotic foci was most prominent in organs of the reticuloendothelial system such as the mesenteric and other lymph nodes and the spleen. In animals from group 4 (200 mg/kg) an increased occurrence of macrophages ("phagocytic cells") with pale and finely granulated cytoplasm was recorded in popliteal and axillary lymph nodes, in the spleen and the thymus. These phagocytic cells were also seen in animals from control and lower dose groups, most frequently in the medullary cords of the lymph nodes, in particular the mesenteric. Areas of chronic necrotizing inflammation without granuloma formation were frequently found in the oral cavity and the nasopharynx, occasionally accompanied by ulceration of the mucous membranes. A number of teeth showed necrotizing pulpitis.
In the liver, granulomatous foci occurred as well as centrilobular necrosis. Occasionally, the hepatic lesion assumed the appearance of recurrent hepatitis with multilobular necrosis, associated with regenerative hyperplasia (described as "nodular hyperplasia").
In the heart, larger areas of necrotizing myocarditis were observed mainly in the subendocardial tissue. The ventricles and/or atria were sometimes dilated by organizing thrombi.
In the renal cortex chronic necrotizing inflammation was seen side by side with lesions of age-related, spontaneous chronic progressive nephropathy. The urinary tract revealed evidence of chronic pyelonephritis and cystitis. Acute tubular necrosis mainly of proximal convoluted tubules near the corticomedullary junction of the kidney was seen in a number of animals of both sexes in the high dose group (200 mg/kg), but also in a few females from the intermediate (30 mg/kg) and low dose (5 mg/kg) group. Focal acute tubular lesions were often associated with the precipitation of crystalline material in proximal convoluted tubules of the same region. Such nephrocalcinosis is a common incidental finding in our colony of female rats. Therefore, the experimental significance of acute tubular necrosis is in doubt.
A number of histological changes most likely resulted from the extensive tissue damage and repair process following chronic necrotizing inflammation. These included cysts and fibrous adhesions of the liver, spleen, peritoneum and kidneys, cystic dilatation of lymphatic vessels, and inflammatory csedema of the peritoneum and mediastinum.
In a number of male (8/19) and female (8/28) rats from group 4 (200 mg/kg) small areas of foam cell accumulations were noted in the ileal mucosa, which otherwise was not affected by inflammatory lesions. In a few cases, chronic necrotizing inflammation occurred in the outer layers of the small intestine. Nine male and two female rats from group 3 (30 mg/kg), which survived the whole treatment period, showed severe ankylosing osteoarthritis (described as "osteoarthrosis", or "chronic inflammation" / "inflammatory cell infiltration" of joints, or as "hypertrophy" of bones) affecting the joints of one or more extremities and/or the tail. The osteoarthritis was occasionally associated with skin ulceration and phlegmonous inflammation. One male control rat and one female rat from group 4 (200 mg/kg) showed comparable changes, however to a lesser extent.
In female groups 3 and 4 (30 and 200 mg/kg) and in male rats from the high dose group (200 mg/kg) there was increased bone marrow cellularity with reduction of the amount of fatty tissue. In group 4 (200 mg/kg) bone marrow hypercellularity appeared to be associated predominantly with an increase of myelopoiesis. In addition, an increased incidence of extramedullary hematopoiesis in lymph nodes was recorded for high dose females (200 mg/kg). Furthermore, extramedullary hematopoiesis of the spleen in these animals was also increased.
Reduced spermatogenesis and atrophy of seminal vesicles in high dose male rats and degenerative lesions of skeletal muscles (myopathy) occurring in high dose (200 mg/kg) rats of either sex was likely due to general debility. Chronic congestion of the liver and oedema of the lung were possibly caused by the extensive chronic necrotizing inflammation prevailing in group 4 (200 mg/kg) as well.
The majority of the macroscopical findings were related to the histological lesions of chronic necrotizing inflammation or they pertained to the other histological findings described above. This also applied to records of enlargement, nodules or masses of the thymus and of the caecum, the histological examination of which revealed chronic necrotizing inflammation of thoracic or mesenteric lymph nodes, respectively.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment with the test article did not produce evidence of oncogenicity. Neither the incidence of any type of tumor nor the total number of primary tumors indicated a treatment-related effect. In contrast, there was evidence for an overall decrease of spontaneously occurring neoplasms in male rats from the high dose group (200 mg/kg) and to a lesser extent also in females from the same dose group. Statistical analysis of the proliferative changes in the adrenal cortex indicated an increased incidence in animals of the high dose group (200 mg/kg) versus the concurrent controls. However, since neoplasms of the adrenal cortex also occur in the untreated rats of our strain, no experimental significance was attached to the occurrence of adenoma or carcinoma found at low incidences in the present study. The frequency of hyperplasia is comparable to the historical control values.
The statistical analysis also indicated a significant occurrence of malignant thymoma in group 4 (200 mg/kg). However, since only one single female (out of 1 surviving females in group 4) was affected, and because benign and malignant thymomas occur in our colony of untreated control rats, this finding was considered incidental without toxicological relevance.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
MIRCOBIOLOGICAL INVESTIGATIONS
In three males Pseudomonas spec, was diagnosed from nose swabs.Two of these also showed Pseudomonas in the trachea. Three males were shown to have E. coli on the tracheal surface. All three females examined showed Staphylococcus from nasal swabs. Except for the above mentioned contamination of the nasal orifices and of air passage surfaces, which were considered irrelevant, no indications of a bacterial disease were found.
The results of the serological investigations indicated a latent but persistent infection with Toolan and/or Kilham viruses. Therefore, in week 83, blood serum of 10 animals each of the control and other treated groups was also subjected to serological investigations. Except for a rare occurrence of syalodacryoadenitis, parainfluenza and Tyzzer's disease the only higher incidence finding was low titers in some animals against Kilham/Toolan. However, the affected animals were distributed among treated and untreated groups such that this finding was considered not to be caused by treatment or to have influenced the outcome of the study.
Details on results:
CLINICAL SIGNS
During the second year of the study a number of males and females from group 200 mg/kg displayed a distended abdomen, piloerection, discoloured eyes in females (reported as "pale"), hunched back and bodyweight loss. These signs generally occurred in the few weeks preceding death. In addition, predominantly in males of groups 30 and 200 mg/kg an increased incidence of swollen hindlimbs was recorded. This was mainly an unilateral finding and for the majority of affected rats was a transient phenomenon.
The lower incidence of palpable masses recorded for males and females of group 4 (200 mg/kg) was related to the early mortality occurring in this group, and hence, the reduced "time at risk " of developing tumours. Among males of group 4 (200 mg/kg) a higher incidence of unilateral eye lesions ocurred, the majority of which resulted from mechanical damage during the withdrawal of blood samples from the orbital sinus . Consequently, ths finding was not considered to be related to treatment.

MORTALITY
Following completion of ten months bf treatment, males and females of group 4 (200 mg/kg) began to die. By week 82 all animals of this group were dead or sacrificed in a moribund condition.
The survival of males of groups 2 and 3 (5 and 30 mg/kg) was similar to that of the control group with 52% and 54% respectively surviving to terminal necropsy compared with 48% of the male controls.
Survival of group 2 and 3 females (5 and 30 mg/kg) was 44 and 42% respectively compared with the survival of 66% in the female control group. However, a number of group 2 females were sacrificed for humane reasons in moribund condition (according to internal standard operating procedures) because of tumors of the mammary gland. The relatively high number of moribund females contributing to the reduced survival of group 2 females was not considered to be related to the treatment for the following reasons:
- the statistical analysis of tumors of the mammary gland indicated no relationship to treatment
- other histopathological findings failed to indicate a common cause of death
- the sacrifice in moribund condition biased the normal mortality
Likewise no biological significance was attributed to the survival analysis of group 3 females, that revealed a statistical significance for increased mortality when compared with the control group. The plot on female mortality reveals a number of obviously premature deaths in groups 2 and 3. However, analysis of these early fatalities (5 and 6 females in groups 2 and 3, respectively which died before week 70) revealed ante mortem / histopathological findings which were all considered not treatment-related.

MIRCOBIOLOGICAL INVESTIGATIONS
In three males Pseudomonas spec, was diagnosed from nose swabs.Two of these also showed Pseudomonas in the trachea. Three males were shown to have E. coli on the tracheal surface. All three females examined showed Staphylococcus from nasal swabs. Except for the above mentioned contamination of the nasal orifices and of air passage surfaces, which were considered irrelevant, no indications of a bacterial disease were found.
The results of the serological investigations indicated a latent but persistent infection with Toolan and/or Kilham viruses. Therefore, in week 83, blood serum of 10 animals each of the control and other treated groups was also subjected to serological investigations. Except for a rare occurrence of syalodacryoadenitis, parainfluenza and Tyzzer's disease the only higher incidence finding was low titers in some animals against Kilham/Toolan. However, the affected animals were distributed among treated and untreated groups such that this finding was considered not to be caused by treatment or to have influenced the outcome of the study.

BODY WEIGHT AND WEIGHT GAIN:
From week 6, lower bodyweight gain for males of group 4 (200 mg/kg) resulted in mean bodyweights significantly lower than controls from week 16 onwards. During the remaining treatment period mean bodyweights of this group remained static.
For the first 6 weeks of treatment positive trends as well as significantly increased mean bodyweights for group 4 females (200 mg/kg) were recorded. These findings are considered incidental since an uneven distribution of mean bodyweights was recorded already for the pretreatment week (week -1). Thereafter, lower bodyweight gains were recorded resulting in significantly lower mean values from week 40 onwards.
Mean bodyweights for males and females of groups 2 and 3 (5 and 3 0 mg/kg) were not influenced by treatment.

FOOD CONSUMPTION:
During the period of weeks 1 to 80, males of group 4 (200 mg/kg) consumed progressively less food than the controls with an overall intake 11 % lower than that of the control group. Females of group 4 (200 mg/kg) however, had food intakes essentially similar to control values apart from a period between weeks 33 and 64 when slightly lower intakes were recorded. The overall intake (weeks 1 - 80) for this group was onl y 4 % lower than the control value. Food intake values for males and females of groups 5 and 30 mg/kg were similar to those of the control group.

FOOD CONSUMPTION RATIOS:
Bodyweight gain was lower than could be expected from lowered diet intake in males and females of group 4 (200 mg/kg). This resulted in higher food consumption ratios during the latter part of this groups survival. Food consumption ratios for other treated groups were similar to those of the respective control groups.

WATER CONSUMPTION:
The water intakes for treated males were similar to those of the control group with overall intakes differing from the control value by less than 3%.
Among treated females, animals of group 2 (5 mg/kg) showed a tendency to consume slightly more water than the controls (overall intake 8.9% higher than the control value). The water intakes for females of groups 3 and 4 (30 and 200 mg/kg) were similar to those of the control group. Therefore, the difference recorded for group 2 was considered of no toxicological relevance.

HAEMATOLOGY:
Among males and females of group 4 (200 mg/kg) treatment produced a slight anemia with hypochromasia and microcytosis associated with higher incidences of anisocytosis, poikilocytosis, and polychromasia of the red blood cells. In addition, a marked leucocytosis occurred with neutrophilia and relative lymphopenia. Hypersegmentation of neutrophils was also noted in most animals. Furthermore, an increase in the number of thrombocytes was recorded for animals of this dose group. To some extent these effects were also noted in animals of group 3 (30 mg/kg).
Differences occurring in other treated groups which attained a level of statistical significance from control values were attributed to spontaneous variation and were considered to be of no clinical relevance.
The lymphatic leukemia detected at terminal laboratory investigation at week 104 in one female of group 3 (30 mg/kg) was not considered treatment-related.

GROSS PATHOLOGY:
The major findings were described as masses, nodules or enlargement of various organs in a number of rats from group 4 (200 mg/kg), primarily of the mesenteric lymph node, which was also affected in some animals from group 3 (30 mg/kg). The other affected tissues included liver, spleen, thymus, salivary gland, heart, adrenal gland, axillary lymph node, kidney and caecum. Some of these organs also showed mottled appearance, fibrinous adhesions and scars.
A number of male and female rats from group 4 (200 mg/kg) presented at autopsy with pleural effusion and ascites, and with oedematous or mottled lungs. Male rats from this dose group showed increased incidence of small seminal vesicles, as well as dilatation of the small intestine.
Some male (9/50) and female (2/50) rats from group 3 (30 mg/kg) which survived the whole test period revealed thickening or hardening of one or more extremities and/or of the tail (ankylosing osteoarthritis). Except for one control male with a thickened hindlimb no such changes were seen in other experimental groups.
All other findings were present in comparable numbers in all experimental groups and were similar to those occurring spontaneously in our colony of rat. Thus no toxicological relevance was attributed to these findings

HISTOPATHOLOGY: NON-NEOPLASTIC:
In animals from groups 3 and 4 (30 and 200 mg/kg) a number of non-neoplastic findings was recorded which consisted of recurrent or chronic inflammatory lesions in various organs. They included circumscribed granulomatous lesions with coagulative necrosis, accompanied by degenerating polymorphs and surrounded by macrophages. The appearance of these cells varied from phagocytes with finely eosinophilic cytoplasm to epitheloid cells or foam cells. Eosinophils were generally scanty and giant cells were rare. Special stains did not give evidence of the presence of acid-fast bacilli or other microorganisms. The size of the lesions ranged from small "initial" granulomas to advanced lesions with necroses extending into the adjacent tissue. A coincidence of small and large lesions at the same site was not uncommon.
The necrotic areas themselves presented frequently as accumulations of densely packed basophilic material suggestive of nuclear debris. The type of lesion described above is termed "chronic necrotizing inflammation" in this report. In some tissues infarct-like necrosis occurred with a lesser degree of cellular reaction. These findings are reported as "necrosis". Chronic necrotizing inflammation was prominent in both sexes from group 4 (200 mg/kg). It occurred most extensively in the mesenteric lymph nodes, affecting all male (47/47) and female (50/50) rats from group 4 (200 mg/kg) and 30/50 males and 18/48 females from group 3 (30 mg/kg). Various other organs were affected by chronic necrotizing inflammation in group 4 (200 mg/kg) rats. The second most frequently involved site in both sexes was the liver, followed by the ovaries in females. Furthermore, frequently involved sites in both sexes were the salivary gland, adrenal gland (incidence ratio between males and females approximately 1:2), spleen, other lymph nodes, teeth (pulp), myocardium, nasopharynx and kidneys. At a low incidence, inflammatory foci were observed at vastly different sites such as mediastinum, peritoneum, pancreas, choroid plexus and brain, adenohypophysis, thyroid gland, skeletal muscle, and vagina. In group 3 (30 mg/kg) chronic necrotizing inflammation was restricted to the mesenteric lymph nodes except for one female rat with additional chronic necrotizing inflammation of the submandibular salivary gland.
Granulomatous reaction associated with the presence of foam cells at the periphery of necrotic foci was most prominent in organs of the reticuloendothelial system such as the mesenteric and other lymph nodes and the spleen. In animals from group 4 (200 mg/kg) an increased occurrence of macrophages ("phagocytic cells") with pale and finely granulated cytoplasm was recorded in popliteal and axillary lymph nodes, in the spleen and the thymus. These phagocytic cells were also seen in animals from control and lower dose groups, most frequently in the medullary cords of the lymph nodes, in particular the mesenteric. Areas of chronic necrotizing inflammation without granuloma formation were frequently found in the oral cavity and the nasopharynx, occasionally accompanied by ulceration of the mucous membranes. A number of teeth showed necrotizing pulpitis.
In the liver, granulomatous foci occurred as well as centrilobular necrosis. Occasionally, the hepatic lesion assumed the appearance of recurrent hepatitis with multilobular necrosis, associated with regenerative hyperplasia (described as "nodular hyperplasia").
In the heart, larger areas of necrotizing myocarditis were observed mainly in the subendocardial tissue. The ventricles and/or atria were sometimes dilated by organizing thrombi.
In the renal cortex chronic necrotizing inflammation was seen side by side with lesions of age-related, spontaneous chronic progressive nephropathy. The urinary tract revealed evidence of chronic pyelonephritis and cystitis. Acute tubular necrosis mainly of proximal convoluted tubules near the corticomedullary junction of the kidney was seen in a number of animals of both sexes in the high dose group (200 mg/kg), but also in a few females from the intermediate (30 mg/kg) and low dose (5 mg/kg) group. Focal acute tubular lesions were often associated with the precipitation of crystalline material in proximal convoluted tubules of the same region. Such nephrocalcinosis is a common incidental finding in our colony of female rats. Therefore, the experimental significance of acute tubular necrosis is in doubt.
A number of histological changes most likely resulted from the extensive tissue damage and repair process following chronic necrotizing inflammation. These included cysts and fibrous adhesions of the liver, spleen, peritoneum and kidneys, cystic dilatation of lymphatic vessels, and inflammatory csedema of the peritoneum and mediastinum.
In a number of male (8/19) and female (8/28) rats from group 4 (200 mg/kg) small areas of foam cell accumulations were noted in the ileal mucosa, which otherwise was not affected by inflammatory lesions. In a few cases, chronic necrotizing inflammation occurred in the outer layers of the small intestine. Nine male and two female rats from group 3 (30 mg/kg), which survived the whole treatment period, showed severe ankylosing osteoarthritis (described as "osteoarthrosis", or "chronic inflammation" / "inflammatory cell infiltration" of joints, or as "hypertrophy" of bones) affecting the joints of one or more extremities and/or the tail. The osteoarthritis was occasionally associated with skin ulceration and phlegmonous inflammation. One male control rat and one female rat from group 4 (200 mg/kg) showed comparable changes, however to a lesser extent.
In female groups 3 and 4 (30 and 200 mg/kg) and in male rats from the high dose group (200 mg/kg) there was increased bone marrow cellularity with reduction of the amount of fatty tissue. In group 4 (200 mg/kg) bone marrow hypercellularity appeared to be associated predominantly with an increase of myelopoiesis. In addition, an increased incidence of extramedullary hematopoiesis in lymph nodes was recorded for high dose females (200 mg/kg). Furthermore, extramedullary hematopoiesis of the spleen in these animals was also increased.
Reduced spermatogenesis and atrophy of seminal vesicles in high dose male rats and degenerative lesions of skeletal muscles (myopathy) occurring in high dose (200 mg/kg) rats of either sex was likely due to general debility. Chronic congestion of the liver and oedema of the lung were possibly caused by the extensive chronic necrotizing inflammation prevailing in group 4 (200 mg/kg) as well.
The majority of the macroscopical findings were related to the histological lesions of chronic necrotizing inflammation or they pertained to the other histological findings described above. This also applied to records of enlargement, nodules or masses of the thymus and of the caecum, the histological examination of which revealed chronic necrotizing inflammation of thoracic or mesenteric lymph nodes, respectively.

HISTOPATHOLOGY: NEOPLASTIC:
Treatment with the test article did not produce evidence of oncogenicity. Neither the incidence of any type of tumor nor the total number of primary tumors indicated a treatment-related effect. In contrast, there was evidence for an overall decrease of spontaneously occurring neoplasms in male rats from the high dose group (200 mg/kg) and to a lesser extent also in females from the same dose group.
Statistical analysis of the proliferative changes in the adrenal cortex indicated an increased incidence in animals of the high dose group (200 mg/kg) versus the concurrent controls. However, since neoplasms of the adrenal cortex also occur in the untreated rats of our strain, no experimental significance was attached to the occurrence of adenoma or carcinoma found at low incidences in the present study. The frequency of hyperplasia is comparable to the historical control values.
The statistical analysis also indicated a significant occurrence of malignant thymoma in group 4 (200 mg/kg). However, since only one single female (out of 1 surviving females in group 4) was affected, and because benign and malignant thymomas occur in our colony of untreated control rats, this finding was considered incidental without toxicological relevance.
Dose descriptor:
NOEL
Remarks:
systemic toxicity
Effect level:
5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
carcinogenicity
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested.
Executive summary:

To determine eventual adverse effects and the potential carcinogenicity of the test compound a carcinogenicity study similar in the design to OECD guideline 451 and in compliance with GLP was conducted. The test compound was administered in the diet at dose levels of 0, 5, 30, and 200 mg/kg bodyweight per day for 24 months to a total of 400 albino rats, 50 males and 50 females per dose group. Average achieved intakes corrected for the quantity of test substance as determined by frequent chemical analysis of the diets were: 4.77, 28.4 and 192 mg/kg bodyweight per day for males and 5.05, 29.2 and 200 mg/kg bodyweight per day for females, of groups 2, 3 and 4 respectively.

Treatment-related clinical signs indicating a general loss of condition (including distended abdomen, hunched posture, discolored eyes, piloerection and bodyweight loss) were confined to males and females of group 4 (200 mg/kg) and occurred in the few weeks preceding death. In addition , swollen hindlimbs were recorded in groups 3 and 4 (30 and 200 mg/kg) . Following 10 months of treatment a progressive mortality occurred among males and females of group 4 (200 mg/kg) with all animals dead or sacrificed by week 82. Treatment resulted in significantly lower bodyweights for males and females of group 4 (200 mg/kg). Bodyweights of other treated groups were not influenced by treatment. Males of group 4 (200 mg/kg) consumed progressively less food whereas the food intakes by group 4 females were essentially similar to control values. The food intake values for other treated groups were similar to control values.Among group 4 rats, higher ratios were obtained during the latter part of the groups survival. Treatment had no effect on the quantity of water consumed. Treatment with 200 mg/kg (group 4) produced a marked leukocytosis with neutrophilia and hyper segmentation of neutrophils. In addition, a slight, hypochromic anemia with microcytosis of red blood cells and a thrombocytosis was noted. To some extent the effects on white blood cells were also seen in animals treated with 30 mg/kg (group 3). Treatment-related macroscopical changes were found in animals from group 3 (30 mg/kg) and group 4 (200 mg/kg). These included enlargement, nodules and masses of various organs in group 4 (200 mg/kg), most frequently of the mesenteric lymph node, which was also affected in group 3 (30 mg/kg), and of some other organs, most notably the liver, spleen and salivary gland. Some male and female rats from group 3 (30 mg/kg) showed ankylosing osteoarthritis affecting the joints of the tail and/or the extremities. In treated animals from groups '3 and 4 (30 and 200 mg/kg) disseminated inflammatory lesions of chronic and recurrent nature, described as "chronic necrotizing inflammation", were found upon microscopical examination. Untreated animals and rats from group 2 (5 mg/kg) were not affected. Inflammation in the tissues included circumscribed granulomatous lesions with coagulative necrosis, as well as neutrophil infiltration and accumulation of foam cells. Chronic necrotizing inflammation occurred most extensively in the mesenteric lymph nodes of all animals from group 4 (200 mg/kg) and in a large number of rats from group 3 (30 mg/kg). In group 3 animals (30 mg/kg) chronic necrotizing inflammation was confined to the mesenteric lymph nodes except for one female rat with the salivary gland affected additionally. In rats from group 4 (200 mg/kg) chronic necrotizing inflammation additionally affected various other organs, most frequently the liver in both sexes and the ovaries in female rats. Furthermore, frequently involved sites in both sexes were the salivary and adrenal gland, the spleen, other lymph nodes, the teeth (with necrotizing pulpitis), the myocardium, the nasopharynx and the kidneys. A smaller incidence of inflammatory foci was also observed at vastly different sites, e.g. mediastinum, peritoneum, chorioid plexus and brain, pancreas, adenohypophysis and vagina. In some rats of either sex from group 4 (200 mg/kg) areas of foam cell accumulation were noted in the ileal mucosa, which otherwise was not affected by inflammatory lesions. Neither the incidence of any type of tumour nor the total number of primary tumours indicated a treatment-related increase.

In conclusion, dietary administration of the test substance at adjusted food concentrations resulting in a dose level of 200 mg/kg bodyweight per day caused reduced longevity in both sexes. Typically, death was preceded by general loss of condition. At 30 and 200 mg/kg bodyweight, histopathological evaluation revealed widespread inflammatory reaction of chronic and recurrent nature, involving principally parts of the reticuloendothelial system, mainly the mesenteric lymph nodes. The hematological, profile indicated leucocytosis with neutrophilia, and additionally in high dose animals thrombocytosis and a slight anemia. Under the conditions of this test, the no effect level is defined as 5 mg/kg bodyweight/day for both sexes. No evidence of carcinogenicity was obtained.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
chronic
Species:
rat

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

No carcinogenicity studies are available. However, from a structurally analogue substance a 24 month feeding study is available.

(See attached document, chapter 13, for read across justification)

Data from the structural analogue:

In a carcinogenicity study similar to OECD guideline 451 and in compliance with GLP the test compound was administered in the diet at dose levels of 0, 5, 30, and 200 mg/kg bodyweight per day for 24 months to a total of 400 albino rats.

Treatment-related clinical signs indicating a general loss of condition (including distended abdomen, hunched posture, discolored eyes, piloerection and bodyweight loss) were confined to males and females of group 4 (200 mg/kg) and occurred in the few weeks preceding death. In addition, swollen hind limbs were recorded in groups 3 and 4 (30 and 200 mg/kg). Following 10 months of treatment a progressive mortality occurred among males and females of group 4 (200 mg/kg) with all animals dead or sacrificed by week 82. Additional findings in the high dose group were significantly lower bodyweights, reduced food consumption in males and elevated food consumption ratios. In hematology, a marked leukocytosis with neutrophilia and hyper segmentation of neutrophils was reported. In addition, a slight, hypochromic anemia with microcytosis of red blood cells and a thrombocytosis was noted. The effects on white blood cells were also seen in animals treated with 30 mg/kg (group 3), although to a lesser extent. Treatment-related macroscopical changes were found in animals from group 3 (30 mg/kg) and group 4 (200 mg/kg). These included enlargement, nodules and masses of various organs in group 4 (200 mg/kg), most frequently of the mesenteric lymph node, which was also affected in group 3 (30 mg/kg), and of some other organs, most notably the liver, spleen and salivary gland. Some male and female rats from group 3 (30 mg/kg) showed ankylosing osteoarthritis affecting the joints of the tail and/or the extremities. In treated animals from groups 3 and 4 (30 and 200 mg/kg) disseminated inflammatory lesions of chronic and recurrent nature, described as "chronic necrotizing inflammation", were found upon microscopical examination. Inflammation in the tissues included circumscribed granulomatous lesions with coagulative necrosis, as well as neutrophil infiltration and accumulation of foam cells. Chronic necrotizing inflammation occurred most extensively in the mesenteric lymph nodes of all animals from group 4 (200 mg/kg) and in a large number of rats from group 3 (30 mg/kg). In group 3 animals (30 mg/kg) chronic necrotizing inflammation was confined to the mesenteric lymph nodes except for one female rat with the salivary gland affected additionally. In rats from group 4 (200 mg/kg) chronic necrotizing inflammation additionally affected various other organs, most frequently the liver in both sexes and the ovaries in female rats. Furthermore, frequently involved sites in both sexes were the salivary and adrenal gland, the spleen, other lymph nodes, the teeth (with necrotizing pulpitis), the myocardium, the nasopharynx and the kidneys. A smaller incidence of inflammatory foci was also observed at vastly different sites, e.g. mediastinum, peritoneum, chorioid plexus and brain, pancreas, adenohypophysis and vagina. In some rats of either sex from group 4 (200 mg/kg) areas of foam cell accumulation were noted in the ileal mucosa, which otherwise was not affected by inflammatory lesions. Neither the incidence of any type of tumor nor the total number of primary tumors indicated a treatment-related increase.

In conclusion, dietary administration of the test substance at a dose level of 200 mg/kg bodyweight per day caused reduced longevity in both sexes. Typically, death was preceded by general loss of condition. At 30 and 200 mg/kg bodyweight, histopathological evaluation revealed widespread inflammatory reaction of chronic and recurrent nature, involving principally parts of the reticuloendothelial system, mainly the mesenteric lymph nodes. The macrophage system was likely of importance. It produced the foam cell reaction visible in the intestine and which was also involved in the extensive inflammatory response in a number of other tissues. Macrophages are well known to be implicated in chronic inflammatory processes which lead to the formation of granuloma and, by the release of a variety of inflammatory mediators, to initiate necrosis or maintain prolonged degenerative conditions. The hematological, profile indicated leucocytosis with neutrophilia, and additionally in high dose animals, thrombocytosis and a slight anemia. Under the conditions of this test, the no effect level is defined as 5 mg/kg bodyweight/day for both sexes. No evidence of carcinogenicity was obtained.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the present data, classification for carcinogenicity is not warranted under Regulation (EC) No.1272/2008.