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Repeated dose toxicity: oral

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chronic toxicity: oral
combined repeated dose and carcinogenicity
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Study conducted according to internationally accepted testing guidelines, well documented and scientifically acceptable. Justification for Read Across is detailed in the Toxicokinetics summary and in the Category Justification Report attached to the section 13.
Reason / purpose:
reference to same study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
equivalent or similar to
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
Pre GLP.
Limit test:

Test material


Test animals

Details on test animals and environmental conditions:
- Source: Winkelmann, Borchen, Germany.
- Age at study initiation: 28 - 32 days old.
- Weight at study initiation: 48 g, average at the start.
- Housing: individually, in Macrolon cages (Type 2).
- Diet: ad libitum, weekly fresh Altromin R-powder feed.
- Water: ad libitum, tap water.

- Temperature: 24 ± 1 °C

Administration / exposure

Route of administration:
oral: feed
unchanged (no vehicle)
Details on oral exposure:
Mixing appropriate amounts with: Altromin R-Pulverfutter (Altromin GmbH, Lage/Lippe, Germany)
Duration of treatment / exposure:
24 months.
Frequency of treatment:
Doses / concentrations
Doses / Concentrations:
0, 100, 1000, 10000 ppm
nominal in diet
No. of animals per sex per dose:
50 males and 50 females per dose.
100 animals in control group.
Control animals:
yes, plain diet


Observations and examinations performed and frequency:
The experimental animals were inspected daily and occurring changes and symptoms recorded.

The body weight of the animals was determined weekly up to 6 months; after that, the weight determination was carried out at intervals of 14 days.

The weekly feed consumption was determined by reweighing.

The blood tests included: determination of the hemoglobin content by cyanhaemiglobin, determination of the hematocrit with a micro-hematocrit centrifuge, calculation of the mean cell volume (MCV) and mean corpuscular hemoglobin content of the (MCH), count of reticulocytes after staining
with brilliant cresyl blue, thrombocytes (platelet) count in a Coulter Counter (model FN), assessment of complete blood count based on smears at 630 times magnification, at end of test determination of the prothrombin time.
The blood sugar determinations were made colorimetrically and enzymatically with glucose oxidase-peroxidase; the cholesterol determination was also performed.

Clinical laboratory investigations were conducted in 5 male and 5 female rats in each dose at 1, 5, 6 and male 12 months; at the end of the experiment, the analysis were conducted to 10 male and female rats.
To test the liver function following enzymes in heparin plasma were determined: Alkaline phosphatase, glutamic-oxaloacetic transaminase and glutamic pyruvic transaminase.

The urine was collected in a 16-hour collection period; the semi-quantitatively determinations of glucose, protein, blood and pH were performed.
Microscopic examinations were performed sediment after centrifugation of urine. For examination of renal function, we determined urea and creatinine.
Sacrifice and pathology:
At the end of the experiment all survivors animals were anesthetized with ether and killed by exsanguination.

The rats dead during the experiment and the rats sacrified at the end were dissected and examined macroscopically.
The weights of the following organs were determined: thyroid, heart, lungs, liver, spleen, kidneys, adrenal glands, testes and ovaries.

The following organs were fixed in Bouin solution, embedded in Paraplast and stained using hematoxylin-eosin (HE): aorta, eyes, small and large intestine, urinary bladder, heart, testis, pituitary gland, salivary gland, liver, lung, lymph nodes, stomach, spleen, epididymis, adrenal glands, kidneys, femur, esophagus, Ovarian, pancreatic, prostate, seminal vesicles, thyroid, skeletal muscle, sternum, trachea, brain, and uterus.
In addition kidney sections were subjected by these rats of the Periodic Acid Schiff (PAS) reaction. The decalcification of the bones was performed in Ethylanadinitrilotetraacetic acid tetrasodium salt (EDTA).
In addition, tumor suspicious changes were evaluated in the same way the histopathological analysis.
The values ​​of the collective test the investigated doses were compared with the control group with the significance test, U-test according to Mann, Whitney and Wilcoxon on the significance level of α = 5 % and α = 1 %.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
comparable with the control group
mortality observed, treatment-related
Description (incidence):
comparable with the control group
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
no significant differences
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
nevertheless the differences from the control are limitated and random
Gross pathological findings:
no effects observed
Description (incidence and severity):
no evidence of treatment related changes in both rats dead before the end of the experiment and the rats killed at the end of the test
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
substance-related histopathological changes were not observed
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
tumors not treatment-related
Details on results:
During the experimental period no differences in appearance and behaviour between treated groups and control were noted. Furthermore no differences in the vividness and coat condition were observed.
Mortality of the dosed groups is comparable to that recorded into the control group.

Comparable in all groups: the bodyweight curve trend of both males and females is graphically superimposable to that traced by the trend of the control group.

No difference from the control group were recorded in food and drink consumption in all dosed groups.

At the 1st month: differences from the control in the hemoglobin and hematocrit content (P < 0.05) were recorded only in the treated males rats groups of doses 100 and 1000 ppm (non dose-dependent); erythrocytes, the number of red blood cells, reticulocytes, white blood cells and platelets, MCH in value and mean cell volume (MCV) were not affected. All the females groups treated did no showed any difference.
At the 3rd month: the only differences from the control were recorded in the erytrocytes and thrombocytes (both P < 0.05) in the male rats group dosed at 1000 ppm.
At the 6th month: a decrease non dose-dependent in the leucocytes count was recorded in the male rats group dosed at 100 ppm (P < 0.05), in comparison with the control group. No difference was recorded in all the other treated groups.
At the 12th month: a decrease in the monocytes (P < 0.05) was observed in the males group dosed at 100 ppm. No other parameter, in no other group was affected.
At the 24th month: at the experiment conclusion only the number of reticulocytes in the male rats dosed at 1000 and 10000 ppm was significantly lower than in the control animals. The rest parameters are missing significant and dose-dependent differences. All the rest of parameters are missing significant and dose-dependent differences. In the analysis of complete blood count, no treatment-related changes in the leukocyte blood count at the dose levels up to 10000 ppm were observed.
Non pathological range blood sugar or cholesterol levels were determined at the dosed groups up to 10000 ppm.

At the 1st month the activity of GOT and GPT transaminases was significantly (P < 0.05) increased in male rats of the dose group of 10000 ppm. In female rats, the GPT activity was significantly (P < 0.01) increased in groups at 1000 and 10000 ppm.
After 3 months of treatment the GOT activity in males is comparable with that in the control, while the GPT trensaminases showed difference in the dosed group at 100 ppm. In all the females groups no differences with the control were recorded.
All the liver function parameters investigated, in all treated groups, at all dosage (both males and females) were comparable to those of the control at the sixth month.
At the 12th month differences non dose-dependent in GOT activity were detected only in the females groups dosed at 100 and 1000 ppm (P < 0.05), while at the 24th months differences in GOT activity from the control were recorded in the male groups treated at 100 and 1000 (P < 0.05 and P < 0.01, respectively) ppm and differences in the total protein determined in serum were recorded in the female group dosed at 10000 ppm ( P < 0.01). Differences in the experiment were not significant.

The findings of the urinalysis after 1 -, 5 -, 6 -, 12 - and 24-month test period revealed no differences between control animals and treated rats up to the 1000 ppm dose. In none of the tested rats glucose, ketone-bodies or bilirubin were found in the urine. Urobilinogen content and pH-value of the treated animals did not differ significantly from the control animals. Positive blood and protein findings were found in approximately equal frequency in the treated as in the untreated rats. The examination of the sediment revealed no treatment-related effect.
The protein contents of the urine were increased in any group.

In comparison to the control group significantly different organ weights were recorded. In male and female rats, the kidney weights were not increased significantly (P < 0.01) up to 10000 ppm. The remaining organ weight differences have to be regarded as limitated and random.

_RATS DEAD during the experiment: no pathological changes which could be attributed to treatment were found in all the animals.
_RATS KILLED at the end of the experiment: no evidence of specific injury in the experimental groups to 10000 ppm

Substance-related histopathological changes were not observed.
In the heart region of the animal 464 (F, 10000 ppm) was found a marked suppurative myocarditis. The animal 359 (F, 1000 ppm) showed pyelonephritis with low-gradiger hyperplasia (Hyper) of the renal pelvic epithelium.
Tumors that would be attributed to the substance were not detected.
_ Cardiovascular and respiratory apparatus: symptoms of chronic inflammation of different degrees were seen in the lungs and tracheae in animals of all groups; nevertheless they are common in old, conventionally held rats.
_Liver: in some animals of different groups of extramedullary hematopoiesis characters (EMH) were seen in the liver in an average small extent. Pathological fatty infiltration were observed in the liver. In many animals bile ducts were altered. Other sporadic effects on singolar animals were seen.
_Genito urinary system: the majority of rats showed renal senile changes of different degrees .In the testes of individuals of different groups atrophy and Spermiogranulome was observed. In the ovaries of many female cysts were not present.
_Adrenal, thyroid and pituitary: in the thyroid and pituitary glands of individual animals of different groups Epithelmeta-neoplasms were observed.
Eyes, intestine, bone, bone marrow, lymph nodes, spleen, pancreas, parotid gland and skeletal muscle showed only treatment-independent individual findings.

In some animals of different groups of extramedullary hematopoiesis characters (EMH) were seen in the liver in an average small extent. Pathological fatty infiltration were observed in the liver.
All tumors detected were considered to be spontaneous, not treatment-related.

Effect levels

open allclose all
Dose descriptor:
Effect level:
709 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: overall effects, highest dose tested
Dose descriptor:
Effect level:
521 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: overall effects, highest dose tested

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Food and active ingredient intake

Dosis [ppm] mean food intake mean active ingredient intake
kg/animal g/animal/day g/kg bw mg/kg bw /day


0 16.48 22.43 - -
100 16.49 22.44 3.84 5.23
1000 16.44 22.36 38.4 52.24
10000 16.61 22.6 382.77 520.78


0 12.5 17.01 - -
100 12.54 17.06 5.16 7.02
1000 12.79 17.4 50.96 69.33
10000 12.86 17.52 521.3 709.25


N. dead / N rat
% N. dead / N rat

1 year

0 2/100 2 0/100 0
100 1/50 2 1/50 2
1000 3/50 6 0/50 0
10000 0/50 0 0/50 0

2 years

0 37/100 37 25/100 25
100 23/50 46 17/50 34
1000 25/50 50 14/50 28
10000 16/50 32 17/50 34

Applicant's summary and conclusion

NOAEL: 709 mg/kg bw/day (actual dose received) (female)
NOAEL: 521 mg/kg bw/day (actual dose received) (male)
Executive summary:


The 50 male and 50 female rats (100 animals in the control group) received test substance administered for 2 years in the following concentrations with the feed: 0 (control), 100, 1000, 10000 ppm.

Appearance, behaviour, feed intake, body weights and mortality were not influenced in male and female animals of doses up to and including 10000 ppm. The animals in the dose groups to 10000 ppm did not show during the entire experimental period any treatment-related symptoms. The growth of the rats was not affected until the dose of 10000 ppm. The haematological investigations performed during and at the end of the test showed no dose of injuries. The significantly lower reticulocyte numbers in the blood of the male rats dosed at 1000 and 10000 ppm after 24 months was not considered as compound-related toxic effect. The studies of male animals to the remaining time points and all investigations in the female animals showed no evidence for a corresponding findings. The clinical chemical analysis, sections and histopathological examinations revealed no evidence for treatment-related damage to the liver. Urinalysis, urea and creatinine concentrations in serum as well as macroscopic and histopathological organ findings did not indicate any influence on the kidneys because the incidence is low (<10%) and the significantly increased kidney weights in male and female rats in the 10000 ppm dose group was not regarded as an expression of injury. Blood sugar and cholesterol levels were not substance-related alterated.
The difference in GOT and GPT activity in male animals after 10000 ppm and GPT activity in the females after 1000 and 10000 pm are not considered signs of liver damage, because all values ​​are in the range of physiological fluctuation band and the investigation into later trial dates did not revealed any findings. The sections of dead and killed rats did not show reference to a specific ending damage that could be caused by the substance. Histopathological examination of the brain, pituitary, aorta, eyes, intestine (duodenum, jejunum, colon), femur, bladder, heart, testis, lung, lymph nodes, stomach, spleen, Nebenhcden, adrenal glands, sciatic nerve, esophagus, parotid gland, ovaries , pancreas, prostate, seminal vesicles, thyroid, skeletal muscle, sternum, thymus, trachea and uterus showed the usual age-related spontaneous alterations. Changes that might be associated with the administration of the substance, not revealed.
From nature, localization, abundance and time of occurrence of the identified benign and malignant tumors was no evidence of a carcinogenic effect of the test item. Thus, the doses of 1000 ppm was well tollerated.