Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 Dec 1994 - 17 Dec 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 Dec 1994 - 17 Dec 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:
adopted 12th May 1981
Deviations:
no
GLP compliance:
yes
Species:
rat
Strain:
Wistar
Remarks:
Hsd/WIN: WU
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 5 - 6 weeks
- Weight at study initiation: 110 - 182 g (males), 102 - 158 g (females)
- Housing: 5 animals separated by sex in Type III Makrolon® and Ha cages, The cages containing the experimental animals were separated by groups and placed on shelves in order of ascending animal number. The position of the shelves was changed on a random basis every 4 weeks.
- Diet: Altromin® 1321 meal (Altromin GmbH, Lage) containing 1% peanut oil, ad libitum except during the urine collection period
- Water: tap water, ad libitum
- Acclimation period: 1 week

DETAILS OF FOOD AND WATER QUALITY:
The tap water complied with drinking water standards in accordance with the Deutsche Trinkwasserverordnung.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 55% ± 5%
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with: the test substance was blended (using a mixing granulator manufactured by Loedige, Paderborn) with Altromin® 1321 containing 1% peanut oil to minimize dust formation (including 0 ppm concentration).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical investigations to demonstrate homogeneity and stability of the test compound in diet preparations were done prior to commencement of the study. The test substance content of diet mixtures fed to the animals was checked analytically at regular intervals throughout the study (start of study, randomly each 3 month period, end of study). Per dose one sample of the food mixes was taken on the day the mixture was prepared, and another was taken after being kept underanimal room conditions for the feeding period (7 days). All these samples were kept deep frozen (at temperatures of approx. -20°C) until analysis.
Duration of treatment / exposure:
24 month
Frequency of treatment:
daily per feed, ad libitum
Dose / conc.:
50 ppm
Remarks:
corresponding to
2.5 mg/kg bw/day (males)
3.4 mg/kg bw/day (females)
Dose / conc.:
200 ppm
Remarks:
corresponding to
10.3 mg/kg bw/day (males)
14.6 mg/kg bw/day (females)
Dose / conc.:
1 000 ppm
Remarks:
corresponding to
52.7 mg/kg bw/day (males)
75.4 mg/kg bw/day (females)
Dose / conc.:
3 000 ppm
Remarks:
corresponding to
170.4 mg/kg bw/day (males)
Dose / conc.:
4 000 ppm
Remarks:
corresponding to
326.7 mg/kg bw/day (females)
No. of animals per sex per dose:
60
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dosages were selected on the basis of the results of a subchronic feeding study in Wistar rats in which the test substance had been administered to 10 males and 10 females at doses of 0, 20, 100, 400, 1600 and 6400 ppm for 13 weeks. In this study concentrations of up to 400 ppm were tolerated without adverse effects. At higher concentrations body weight depression, effects on blood parameters (essentially reduced hemoglobin and hematocrit values and elevated reticulocyte means), inhibition of the cholinesterase activity (erythrocytes) and signs of changed liver function such as altered biochemical parameters (enhanced cholesterol plasma levels, reduced contents of triglycerides and unesterified fatty acids, lower albumin means), enzyme induction, increased liver weights and occurrence of hepatocellular hypertrophy were evident. Rats receiving 6400 ppm exhibited diffuse hyperplasia of the bladder urothelium as well. In further electron-microscopical investigations on the urinary bladder cytotoxicity with mild regenerative hyperplasia was detected in 6400 ppm males (females were not investigated) with higher frequency than in controls.

- Rationale for animal assignment: The rats were weighed individually beforehand and the required number of animals were then grouped by weight (light, midde and heavy subgroups) and randomly distributed into large containers.
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
twice daily and daily at weekends and on bank holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
Parameters: orifices, posture, general behavior, respiration and excretory products were carefully examined, with any significant findings were registered. Animals that became ill or had developed neoplasms that could lead to death were kept apart, observed more frequently, and killed prematurely if death was imminent.

BODY WEIGHT: Yes
- Time schedule for examinations:
weekly from start to Week 13, and every 2 weeks thereafter up to Week 105

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: weekly within the first 13 weeks of treatment and every 4 weeks thereafter up to Week 101

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations and dose groups that were examined:
during the first two weeks of the study in all rats. In Week 54 and prior to the final bleeding all living animals scheduled for the final necropsy from the control group and the 3000 or 4000 ppm dose groups were examined. At study termination 200 and 1000 ppm males were investigated, additionally.

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
Weeks 26/27, 52, 78 and 105/106
- Anaesthetic used for blood collection: Yes, for glucose and deproteinized whole blood only
- Animals fasted: Yes
- How many animals:
10
- Parameters analysed: differential blood count, erythrocyt morphology, erythrocyte count, Heinz bodies, hemoglobin, hematocrit, leucocyte count, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, platelet count, thromboplastin time, reticulocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Weeks 26/27, 52, 78 and 105/106
- Animals fasted: Yes
- How many animals:
10
- Parameters analysed: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, glutamate dehydrogenase, glucose, bilirubin, albumin, cholesterol, creatinine, total protein, triglyceride, urea, calcium, sodium, potassium, chloride and inorganic phosphate

URINALYSIS: Yes
- Time schedule for collection of urine:
Weeks 26/27, 52, 79 and 98/99
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters analysed: sediment, bilirubin, blood, glucose, ketone bodies, pH, protein, urobilinogen (semiquantitative), density, volume, total protein (quantitative)

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER:
Cholinesterase activity in brain
- Time schedule: Weeks 52/53 and 104/105

Cholinesterase activities in plasma and erythrocytes
- Time schedule: Week 26/27, measurement were repeated for males in the following week due to some implausible values

Determination of crystals in urine sediment
- Time schedule: Week 103
- Dose groups that were examined: 8 animals, in controls and rats of the 3000 and 4000 ppm groups
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Organ weights: brain, heart, liver, spleen, kidneys, adrenals, testes, ovaries

HISTOPATHOLOGY: Yes, the following organs and tissues were fixed for analysis: Adrenals , Aorta , Brain (3 locations) [in some cases half brain] , Caecum, Colon, Duodenum, Epididymides , Esophagus, Eyes (with eyelids), Exorbital lacrimal glands, Femur (incl. bone marrow and knee joint), Harderian glands, Head-Nose-Pharynx area, Heart, lleum, Jejunum, Kidneys, Larynx, Liver, Lungs, Lymph nodes (mandibular and mesenteric), Mammary glands, Optic nerve, Ovaries (incl. oviduct), Pancreas, Pituitary, Prostate, Rectum, Residual Intestine, Salivary glands, Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (3 regions), Spleen, Sternum (with bone marrow), Stomach (with forestomach), Tattooed ears, Testes, Thymus (if present), Thyroid (parathyroid), Tongue, Trachea, Ureters, Urethra, Urinary bladder, Uterus, Vagina, Zymbal glands and all tissues showing abnormalities
Statistics:
Dunnett-Test in connection with a variance analysis (body and organ weight data)

Analysis of variance followed by Dunnett test (Erythrocytes, Hemoglobin, Hematocrit, Mean Corpuscular Hemoglobin, Mean Corpuscular Hemoglobin Concentration, Mean Corpuscular Volume Erythrocytes, Thrombocytes, Albumin, Creatinine, Chloride, Glucose, Protein, Triglycerides, Urea)

Kruskal-Wallis-Test with a Steel-Test (food and water intake data)

Kruskal-Wallis test followed by adjusted U test (Heinz Bodies, Hepato Quick, Leucocytes, Reticulocytes, Band NeutrophiIs, EosinophiIs, Lymphocytes, Monocytes, Segmented Neutrophils, Bilirubin total, Gamma-Gtutamyltransferase, Specific Gravity, Glucose, dipstick, pH, dipstick, Protein Quantitative, Volume, pH)

Adjusted Welsh test (Atanine aminotransferase, Alkaline phosphatase, Aspartate aminotransferase, Choiinesterase, Choiinesterase in Erythrocytes, Choiinesterase in Brain, Cholesterol, Calcium, Glutamate dehydrogenase, Potassium, Sodium, Inorganic Phosphate)
Clinical signs:
no effects observed
Description (incidence and severity):
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
All dose groups: a higher mortality in females was evident from Week 52 onwords; dose correlation is lacking

For details please refer to Table 1 in "any other information on results incl. tables".
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
3000/4000 ppm: body weight depression in males from Week 7 / in females from Week 13 onwords

For details please refer to Table 2 in "any other information on results incl. tables".
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
3000/4000 ppm: slightly elevated food intake per kg body weight in both sexes (12.3%/19.2%)
The test substance intake in the treatment groups roughly corresponds to the theoretical dose intervals.

For details please refer to Table 3 in "any other information on results incl. tables".
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
3000/4000 ppm: indication for slightly reduced food efficiency in both sexes

For details please refer to Table 3 in "any other information on results incl. tables".
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
no effects observed

For details please refer to Table 4 in "any other information on results incl. tables".
Ophthalmological findings:
effects observed, treatment-related
Description (incidence and severity):
1000 and 3000 ppm: higher incidence of moderate or severe opacities in the (whole) lens cortex of males at termination

For details please refer to Table 5 in "any other information on results incl. tables".
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
3000/4000 ppm: decreased Hb and Ht in males in Week 26/27 only, decreased MCH and MCHC in females

For details please refer to Tables 6 and 7 in "any other information on results incl. tables".
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
3000/4000 ppm: slightly increased gamma glutamyltransferase (GOT) activity in both sexes), but not at all dates and without relevant time-dependent increase
Slightly decreased ASAT and APh activities in animals dosed at 1000 ppm and higher are not considered to indicate an adverse effect, since the values were within the physiological range.

For details please refer to Table 8 in "any other information on results incl. tables".
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Quantitative urinalyses did not reveal a treatment-related effect on pH, sediment, proteinurea, urine volume or urine density. Significant deviations from control values in these parameters were not considered relevant, since dose correlations were lacking.

For details please refer to Tables 10 and 11 in "any other information on results incl. tables".
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):
Interim necropsy:
3000/4000 ppm: significantly increased liver weights (absolute and relative ) in both sexes
Terminal necropsy:
3000/4000 ppm: partly significantly increased liver weights (absolute and relative) in both sexes, slightly decreased spleen weights (absolute and relative) in both sexes, increased relative testis weights in males

For details please refer to Tables 12 and 13 in "any other information on results incl. tables".
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Interim necropsy:
no effects observed
Terminal necropsy:
3000/4000 ppm: 6 males and 5 females appeared skinny
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Interim examinations:
200 ppm: diffuse hyperplasia in the transitional epithelium of the urinary bladder in males
1000 ppm: diffuse hyperplasia in the transitional epithelium of the urinary bladder in males, hepatocellular hypertrophy (centrilobular) in both sexes
3000/4000 ppm: diffuse hyperplasia in the transitional epithelium of the urinary bladder, hepatocellular hypertrophy (centrilobular) and slight increase in the incidence and severity of vacuolation of zona fasciculata cells in the adrenal cortex in both sexes

For details please refer to Table 14 in "any other information on results incl. tables".

Terminal examinations:
1000 ppm: centrilobular hepatocytes with cytoplasmic changes and/or hypertrophy more frequently and decreasing tendency for pituitary hyperplasia as well as for degenerative alterations in the adrenals, tongue, heart and kidney in females
3000/4000 ppm: eosinophilic foci, focal degenerative changes (periportal) and cytoplasmic changes of hepatocytes (periportal and centrilobular) in the liver of males, peliotic foci, single cell necrosis and cytoplasmic changes of hepatocytes (centrilobular) and/or hypertrophy in the liver of females;
increased number of animals with a focal or diffuse simple transitional hyperplasia in the urinary bladder, increases of Proliferating Cell Nuclear Antigen (PCNA) labelling index of the urinary bladder in the hyperplastic segment of the transitional epithelium of females;
increased incidence of follicular hyperplasia (males) and mineralization of the follicular colloid (both sexes) in thyroid glands;
increased number of males with increase in vacuolation of cells in the zona fasciculata of the adrenal glands;
higher frequency of atrophy and/or fibre degenerations of the skeletal muscle in females;
markedly higher incidence and degree of degenerative myelinopathy in the sciatic nerve in both sexes;
decreasing tendency for pituitary hyperplasias as well as for degenerative alterations in the adrenals, tongue, heart and kidney in females;
dilated glands in the stomach mucosa and increase in pigment storage in the spleen of females

For details please refer to Table 15 in "any other information on results incl. tables".
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Interim examinations:
In animals scheduled for interim kill, a no dose-related occurrence of a tumour type was evident.
Terminal examinations:
1000 ppm: adenomas in the thyroid follicles of one male
3000/4000 ppm: one transitional cell carcinoma and two transitional cell papillomas in the urinary bladder of females and one transitional carcinoma in the urethra of a male;
adenomas/carcinoma in the thyroid follicles of males (incidence per increasing dose group: 0-0-0-1/0-2/1)
For details please refer to Tables 16 to 19 in "any other information on results incl. tables".
Other effects:
effects observed, treatment-related
Description (incidence and severity):
ChE activity plasma
No relevant treatment-related effects observed.

ChE activity erythrocytes
1000 ppm: significantly reduced ChE activity in both sexes
3000/4000 ppm: significantly reduced activity in both sexes

ChE activity brain
1000 ppm: reduced ChE activity in females
3000/4000 ppm: reduced ChE activity in both sexes

For details please refer to Table 9 in "any other information on results incl. tables".
Details on results:
Mortality:
The number of males which had died unscheduled was equally distributed among the groups up to 3000 ppm. In all female treatment groups a higher mortality is evident from week 52 onwards. Since a dose correlation is lacking in the wide dose range (50 and 4000 ppm) a treatment effect is not assumed. Neither for males nor for females did the global comparison of the survival curves (Wilcoxon Test) gave any indication of statistically relevant differences between the groups (p = 0.5782).

Body weight and weight changes:
There were no significant and dosedependent effects on body weights up to 1000 ppm in males. Males ingesting 3000 ppm gained less body weight from week 7 onwards with a maximum difference of 11.0% in week 105. The differences were significant in most cases. In female rats there is also no toxicologially relevant effect on body weights up to the dose of 1000 ppm. The few significantly lower means at 1000 ppm (between weeks 43 to 61) are not interpreted as an adverse effect, since deviations to control data are very small (maximal 6.3% in week 51). At 4000 ppm a significant (from week 13 onwards) body weight depression was observed with a maximum of 14.3% in week 85.

Food consumption and compound intake:
The food intake per animal at doses of up to 3000 ppm (males) or 4000 ppm (females) was comparable to that of untreated rats. The slightly elevated intake data per kg body weight in rats receiving 3000 (12.3%) or 4000 ppm (19.2%) could be interpreted as an indication of a slightly reduced feed efficiency.

Opthalmological findings:
Males of the 1000 and 3000 ppm groups showed a higher incidence of moderate or severe opacities in the (whole) lens cortex. The highest frequency of lenses showing slight (wedge shaped) waterclefts, being a prestage of cortical opacities, was noted in 0 ppm males. This incidence distribution indicates a shift of lens waterclefts towards cortical opacities in 1000 and 3000 ppm males. In 4000 ppm females no remarkable lens findings could be detected compared to controls. Concerning the remaining eye compartments no treatment effect is visible.

Haematological findings:
There were no toxicologically relevant changes in the erythrocyte parameters (count, MVC, MCHC and MCH), in the hemoglobin concentration or in the hematocrit in males or females up to 1000 ppm. Some significant differences in these groups and parameters are not considered as a sign of toxicity, since deviations to control values were very small (MCV, MCH and elevated MCHC means in treated males in week 26/27) and/or a dose correlation was lacking (erythrocyte counts). The hemoglogin concentration and hematocrit values were significantly reduced in 3000 males at week 26/27, but not thereafter. Other significant changes in this group were slight and without corroborating findings. In 4000 ppm females no significant changes were seen with regard to hemoglobin concentration, hematocrit and erythrocyte counts but the calculation of MCH and MCHC revealed consistently lower (partly p<0.01) values than in controls and remaining groups. Analysis of erythrocyte morphology did not reveal abnormalities that could be regarded as a toxic effect. No relevant changes in platelet counts were found in treated males and females. The very slight increase in the means of platelet count of 4000 ppm females in week 26/27 and 52 do not reflect an adverse effect, since individual values were within the reference range. The means of the thromboplastin time in the treatment groups were not remarkably different from those at 0 ppm. No changes of reticulocytes was observed up to highest dose level in males and females. No toxicologically relevant changes in leukocyte counts and differential blood counts were visible in the dose range investigated.

Clinical biochemistry findings:
Neither male nor female rats showed any remarkable changes in the plasma activity of aspartate aminotransferase (ASAT), alkaline phosphatase (APh) and glutamate dehydrogenase (GLDH) up to 200 ppm. At higher doses there was a tendency towards slightly (sometimes significantly) lower aspartate aminotransferase (ASAT) and alkaline phosphatase (APh) activities. These changes were not considered to indicate an adverse effect, since the values were within the physiological range. The activity of the alanine aminotransferase (ALAT) was unchanged in all treatment groups. A trend towards slightly elevated gamma glutamyltransferase (GOT) activity was at 3000 or 4000 ppm (p<0.01), but not at all dates and without relevant time dependent increase.

Urinalysis findings:
In week 98/99 quantitative pH measurements were performed by a microelectrode. Results of these determinations do not indicate any influence of the test substance on the urine pH in the dose range investigate. Scanning-electron-microscopy of urine sediment did not reveal any indication of a change in amount or morphology of urine crystals due to the treatment. Quantitative urinalyses did not reveal a treatment-related effect on proteinurea, urine volume or urine density. Significant deviations from control values in these parameters were not considered relevant, since dose correlations were lacking.

Organ weight findings including organ body weight ratios:
At interim necropsy all absolute and relative organ weights except the liver weights were comparable with those of controls. A few significant changes can be attributed to differences in body weight. The liver weights were not remarkably changed up to 1000 ppm. In 3000 ppm males they were significantly increased by 18.5% (absolute) and 17.6% (relative), whereas in 4000 ppm females the increase was 18.8% (absolute) and 41.7% (relative). At terminal necropsy there were no toxicologically relevant differences between the dose groups and the controls with respect to the weights of the brain, adrenals, heart, kidneys or ovaries. The few differences marked as significant in these organs were very small and attributable to varying body weights. The liver weights were inconspicuous up to 1000 ppm. At higher concentrations absolute and relative liver weights were increased (partly p<0.01). The relative means were 20.2% ( 3000 ppm males) or 32.9% (4000 ppm females) above the corresponding control value. Up to 1000 ppm spleen and testis weights were unremarkable. At the top concentration levels slightly lower (up to -23%) spleen weights (absolute and relative) were seen in both sexes and the relative testes weights were elevated (p<0.01) by about 17%. These deviations were partly significant.

Gross pathological findings:
No treatment related macroscopical findings were observed in rats scheduled for a treatment period of 12 months. Gross-pathological findings recorded at the necropsies performed during the in life phase in case of unscheduled deaths or at the end of the study revealed no evidence of dose-related organ lesions in any of the groups up to and including doses of 3000 or 4000 ppm. There were six 3000 ppm males and five 4000 ppm females that appeared skinny at necropsy.

Histpathological findings (non neoplastic):
In animals scheduled for a 12-months treatment revealed test substance-related lesions in the adrenals, liver and urinary bladder. In the adrenal cortex a slight increase in the incidence and severity of vacuolation of the zona fasciculata cells was recorded at 3000 or 4000 ppm. A minimal to moderate hepatocellular hypertrophy, predominantly centrolobular, was noted in rats at 1000 ppm and above.
In the urinary bladder a diffuse hyperplasia (minimal to slight) was observed in the transitional epithelium at 200 ppm and above (males) or in the group 4000 ppm (females). For the lesions in the urinary bladder, liver and adrenals a statistically significant trend was established. All other microscopical findings recorded were considered to be incidental. In animals scheduled for terminal kill, there were no treatment-related non-neoplastic lesions at concentrations of up to 1000 ppm in males and 200 ppm in females. In the liver of 3000 ppm males eosinophilic foci, focal degenerative changes (periportal) and cytoplasmic changes of hepatocytes (periportal and centrilobular) were noted with a markedly higher incidence than in the other groups. These findings were associated with a reduced number of clear cell foci at 3000 ppm. In males of the groups 0, 50, 200 and 1000 ppm and in all treated females the frequency of all these findings did not exceed the reference range of rats of this age. In females centrilobular hepatocytes with cytoplasmic changes and/or hypertrophy were detected more frequently from 1000 ppm onwards. Additionally, in 4000 ppm females peliotic foci and single cell necroses were noted with increased incidences. Most of the liver lesions mentioned were found to show a statistically significant positive trend. The number of animals with a focal or diffuse simple transitional hyperplasia in the urinary bladder was increased (trend mostly p<0.01) in high dose males and females. In 4000 ppm females atrophy (p<0.01) and/or fibre degenerations (p<0.05) of the skeletal muscle were found with a higher frequency than in the other groups. In 3000 ppm males and 4000 ppm females a degenerative myelinopathy was detected in the sciatic nerve with a markedly higher incidence (trend p<0.01) and a higher degree than in the other groups, where the frequency of this lesion did not exceed the range normally found in old rats. In the thyroid glands the incidence of follicular hyperplasia (males) and mineralization of the follicular colloid (males and females) was significantly increased in 3000/4000 ppm rats. At 3000 ppm there were more (trend p<0.01) males exhibiting an increase in vacuolation of cells in the zona fasciculata of the adrenal glands. Statistically significant increases of proliferating cell nuclear antigen (PCNA) labeling index of the urinary bladder were only achieved in the hyperplastic segment of the transitional epithelium of 4000 ppm females. In the bladder of 1000 ppm females and in the non-hyperplastic bladder segment of the 4000 ppm rats the calculated values were similar to those of control females.The remaining observed findings were considered to be secondary possibly due to retarded aging, which is often seen in toxicological studies, if body weight is reduced as in the present study. These findings included a decreasing tendency for pituitary hyperplasias as well as for degenerative alterations in the adrenals, tongue, heart and kidney essentially in 1000 and 4000 ppm females. The elevated (p<0.05) number of 4000 ppm females showing dilated glands in the stomach mucosa was considered unspecific and not treatment-related. Also the increase in pigment storage in the spleen (p<0.01) was not considered treatment-related.

Histpathological findings (neoplastic):
In animals scheduled for interin kill, a no dose-related occurrence of a tumor type was evident. At the terminal kill, one transitional cell carcinoma and two transitional cell papillomas (trend p<0.01) were detected in the urinary bladder of 4000 ppm females, whereas no bladder tumor occurred in the remaining female groups or in males. Additionally, one transitional carcinoma was seen in the urethra of a 3000 ppm male. In thyroid follicles of males adenomas were observed with a frequency of 0-0-0-1-2 (trend p<0.05) and carcinomas with an incidence of 0-0-0-0-1. Uterine adenocarcinomas were noted in 4000 ppm females more often (trend p<0.01) than in the other groups. Additionally, there was one squamous cell carcinoma (p<0.05) in this group. However, the incidences of both tumor types do not exceed the historical control range and are therefore, considered to be not compound-induced. Decreased tumor incidences were noted for the mammary fibroadenomas (15-10-7-6-5) and pituitary adenomas (24-18-24-14-11) in the female treatment groups. The incidences of the remaining tumors were not distributed in a treatment-related manner and comparable with those known from own historical control collectives.The number of cases, in which a tumor type occurred in one group only was comparable in the 3000/4000 ppm and 0 ppm group (Please refer to table 16 in the "any other information on results incl. tables " section). The overall tumor incidence and the total number of benign and malignant neoplasms per group were not increased in treated males and females at all concentrations (Please refer to table 17 in the "any other information on results incl. tables " section). The number of malignant tumors was even reduced in males receiving 200 ppm and above. No remarkable differences were observed deaths in the number of tumor bearing rats or animals with a benign, malignant or benign and malignant tumor between the control and treatment groups for both, intercurrent or scheduled deaths (Please refer to table 18 in the "any other information on results incl. tables " section). As regards the time of occurrence of the tumors, there were no significant differences between treated rats and controls (Please refer to table 19 in the "any other information on results incl. tables " section).

Other findings: Cholinesterase activities (CHE)
No significant CHE inhibition occurred in the plasma. The 24% lower plasma CHE activity of 3000 ppm males (week 78) is not regarded as toxicologically relevant, because this was an isolated event, where in the control group relatively high activities were measured compared to those of the other time points. In addition, plasma CHE activity was even not affected when e.g. CHE activity in erythrocytes was inhibited by 90%. No remarkable CHE inhibition in erythrocytes were detected in males and females up to 200 ppm. At higher concentrations a dose-dependently and significantly lower CHE activity (inhibited up to 82% in males and 90% in females) was measured compared to control means. The significantly lower activity in 200 ppm females in week 52/53 is considered to be incidental, because activity at this dose level was not remarkably inhibited at the other three time points, where stronger effects were noted in the two highest dose groups. Also in week 52/53, a relatively high activity in controls may have contributed. Up to 3000 ppm (males) and 200 ppm (females) no significant or biologically relevant effect was noted on the brain CHE activity. In females there was a significantly reduced activity at 1000 and 4000 ppm (up to 41%). The inhibition values of 13 and 17% in 1000 ppm females and those of 14% calculated for 3000 ppm males at termination might be of questionable toxicological relevance.
Relevance of carcinogenic effects / potential:
Mortality:
The number of males which had died unscheduled was equally distributed among the groups up to 3000 ppm. In all female treatment groups a higher mortality is evident from Week 52 onwards. Since a dose correlation is lacking in the wide dose range (50 and 4000 ppm) a treatment effect is not assumed. Neither for males nor for females did the global comparison of the survival curves (Wilcoxon Test) gave any indication of statistically relevant differences between the groups (p = 0.5782).

Body weight and weight changes:
There were no significant and dose-dependent effects on body weights up to 1000 ppm in males. Males ingesting 3000 ppm gained less body weight from week 7 onwards with a maximum difference of 11.0% in Week 105. The differences were significant in most cases. In female rats there is also no toxicologically relevant effect on body weights up to the dose of 1000 ppm. The few significantly lower means at 1000 ppm (between Weeks 43 to 61) are not interpreted as an adverse effect, since deviations to control data are very small (maximal 6.3% in Week 51). At 4000 ppm a significant (from Week 13 onwards) body weight depression was observed with a maximum of 14.3% in Week 85.

Food consumption and compound intake:
The food intake per animal at doses of up to 3000 ppm (males) or 4000 ppm (females) was comparable to that of untreated rats. The slightly elevated intake data per kg body weight in rats receiving 3000 (12.3%) or 4000 ppm (19.2%) could be interpreted as an indication of a slightly reduced feed efficiency.

Opthalmological findings:
Males of the 1000 and 3000 ppm groups showed a higher incidence of moderate or severe opacities in the (whole) lens cortex. The highest frequency of lenses showing slight (wedge shaped) waterclefts, being a pre-stage of cortical opacities, was noted in control males. This incidence distribution indicates a shift of lens waterclefts towards cortical opacities in 1000 and 3000 ppm males. In 4000 ppm females no remarkable lens findings could be detected compared to controls. Concerning the remaining eye compartments no treatment effect is visible.

Haematological findings:
There were no toxicologically relevant changes in the erythrocyte parameters (count, MVC, MCHC and MCH), in the haemoglobin concentration or in the haematocrit in males or females up to 1000 ppm. Some significant differences in these groups and parameters are not considered as a sign of toxicity, since deviations to control values were very small (MCV, MCH and elevated MCHC means in treated males in Week 26/27) and/or a dose correlation was lacking (erythrocyte counts). The haemoglobin concentration and haematocrit values were significantly reduced in 3000 males at Week 26/27, but not thereafter. Other significant changes in this group were slight and without corroborating findings. In 4000 ppm females no significant changes were seen with regard to haemoglobin concentration, haematocrit and erythrocyte counts but the calculation of MCH and MCHC revealed consistently lower (partly p<0.01) values than in controls and remaining groups. Analysis of erythrocyte morphology did not reveal abnormalities that could be regarded as a toxic effect. No relevant changes in platelet counts were found in treated males and females. The very slight increase in the means of platelet count of 4000 ppm females in Weeks 26/27 and 52 do not reflect an adverse effect, since individual values were within the reference range. The means of the thromboplastin time in the treatment groups were not remarkably different from those at 0 ppm. No changes of reticulocytes were observed up to the highest dose level in both sexes. No toxicologically relevant changes in leukocyte counts and differential blood counts were visible in the dose range investigated.

Clinical biochemistry findings:
Neither male nor female rats showed any remarkable changes in the plasma activity of aspartate aminotransferase (ASAT), alkaline phosphatase (APh) and glutamate dehydrogenase (GLDH) up to 200 ppm. At higher doses there was a tendency towards slightly (sometimes significantly) lower aspartate aminotransferase (ASAT) and alkaline phosphatase (APh) activities. These changes were not considered to indicate an adverse effect, since the values were within the physiological range. The activity of the alanine aminotransferase (ALAT) was unchanged in all treatment groups. A trend towards slightly elevated gamma glutamyltransferase (GOT) activity was at 3000 or 4000 ppm (p<0.01), but not at all dates and without relevant time dependent increase.

Urinalysis findings:
In Week 98/99 quantitative pH measurements were performed by a microelectrode. Results of these determinations do not indicate any influence of the test substance on the urine pH in the dose range investigated. Scanning-electron-microscopy of urine sediment did not reveal any indication of a change in amount or morphology of urine crystals due to the treatment. Quantitative urinalyses did not reveal a treatment-related effect on proteinurea, urine volume or urine density. Significant deviations from control values in these parameters were not considered relevant, since dose correlations were lacking.

Organ weight findings including organ body weight ratios:
At interim necropsy all absolute and relative organ weights except the liver weights were comparable with those of controls. A few significant changes can be attributed to differences in body weight. The liver weights were not remarkably changed up to 1000 ppm. In 3000 ppm males they were significantly increased by 18.5% (absolute) and 17.6% (relative), whereas in 4000 ppm females the increase was 18.8% (absolute) and 41.7% (relative). At terminal necropsy there were no toxicologically relevant differences between the dose groups and the controls with respect to the weights of the brain, adrenals, heart, kidneys or ovaries. The few differences marked as significant in these organs were very small and attributable to varying body weights. The liver weights were inconspicuous up to 1000 ppm. At higher concentrations absolute and relative liver weights were increased (partly p<0.01). The relative means were 20.2% ( 3000 ppm males) or 32.9% (4000 ppm females) above the corresponding control value. Up to 1000 ppm spleen and testis weights were unremarkable. At the top concentration levels slightly lower (up to -23%) spleen weights (absolute and relative) were seen in both sexes and the relative testes weights were elevated (p<0.01) by about 17%. These deviations were partly significant.

Gross pathological findings:
No treatment-related macroscopical findings were observed in rats scheduled for a treatment period of 12 months. Gross-pathological findings recorded at the necropsies performed during the in life phase in case of unscheduled deaths or at the end of the study revealed no evidence of dose-related organ lesions in any of the groups up to and including doses of 3000 or 4000 ppm. There were six 3000 ppm males and five 4000 ppm females that appeared skinny at necropsy.

Histopathological findings (non-neoplastic):
In animals scheduled for a 12-months treatment revealed test substance-related lesions in the adrenals, liver and urinary bladder. In the adrenal cortex a slight increase in the incidence and severity of vacuolation of the zona fasciculata cells was recorded at 3000 or 4000 ppm. A minimal to moderate hepatocellular hypertrophy, predominantly centrolobular, was noted in rats at 1000 ppm and above.
In the urinary bladder a diffuse hyperplasia (minimal to slight) was observed in the transitional epithelium at 200 ppm and above (males) or in the group 4000 ppm (females). For the lesions in the urinary bladder, liver and adrenals a statistically significant trend was established. All other microscopical findings recorded were considered to be incidental. In animals scheduled for terminal kill, there were no treatment-related non-neoplastic lesions at concentrations of up to 1000 ppm in males and 200 ppm in females. In the liver of 3000 ppm males eosinophilic foci, focal degenerative changes (periportal) and cytoplasmic changes of hepatocytes (periportal and centrilobular) were noted with a markedly higher incidence than in the other groups. These findings were associated with a reduced number of clear cell foci at 3000 ppm. In males of the groups 0, 50, 200 and 1000 ppm and in all treated females the frequency of all these findings did not exceed the reference range of rats of this age. In females centrilobular hepatocytes with cytoplasmic changes and/or hypertrophy were detected more frequently from 1000 ppm onwards. Additionally, in 4000 ppm females peliotic foci and single cell necrosis were noted with increased incidences. Most of the liver lesions mentioned were found to show a statistically significant positive trend. The number of animals with a focal or diffuse simple transitional hyperplasia in the urinary bladder was increased (trend mostly p<0.01) in high dose males and females. In 4000 ppm females atrophy (p<0.01) and/or fibre degenerations (p<0.05) of the skeletal muscle were found with a higher frequency than in the other groups. In 3000 ppm males and 4000 ppm females a degenerative myelinopathy was detected in the sciatic nerve with a markedly higher incidence (trend p<0.01) and a higher degree than in the other groups, where the frequency of this lesion did not exceed the range normally found in old rats. In the thyroid glands the incidence of follicular hyperplasia (males) and mineralization of the follicular colloid (males and females) was significantly increased in 3000/4000 ppm rats. At 3000 ppm there were more (trend p<0.01) males exhibiting an increase in vacuolation of cells in the zona fasciculata of the adrenal glands. Statistically significant increases of proliferating cell nuclear antigen (PCNA) labeling index of the urinary bladder were only achieved in the hyperplastic segment of the transitional epithelium of 4000 ppm females. In the bladder of 1000 ppm females and in the non-hyperplastic bladder segment of the 4000 ppm rats the calculated values were similar to those of control females.The remaining observed findings were considered to be secondary possibly due to retarded aging, which is often seen in toxicological studies, if body weight is reduced as in the present study. These findings included a decreasing tendency for pituitary hyperplasia as well as for degenerative alterations in the adrenals, tongue, heart and kidney essentially in 1000 and 4000 ppm females. The elevated (p<0.05) number of 4000 ppm females showing dilated glands in the stomach mucosa was considered unspecific and not treatment-related. Also the increase in pigment storage in the spleen (p<0.01) was not considered treatment-related.

Histpathological findings (neoplastic):
In animals scheduled for interim kill, a no dose-related occurrence of a tumour type was evident. At the terminal kill, one transitional cell carcinoma and two transitional cell papillomas (trend p<0.01) were detected in the urinary bladder of 4000 ppm females, whereas no bladder tumour occurred in the remaining female groups or in males. Additionally, one transitional carcinoma was seen in the urethra of a 3000 ppm male. In thyroid follicles of males adenomas were observed with a frequency of 0-0-0-1-2 (trend p<0.05) and carcinomas with an incidence of 0-0-0-0-1. Uterine adenocarcinomas were noted in 4000 ppm females more often (trend p<0.01) than in the other groups. Additionally, there was one squamous cell carcinoma (p<0.05) in this group. However, the incidences of both tumour types do not exceed the historical control range and are therefore, considered to be not compound-induced. Decreased tumour incidences were noted for the mammary fibroadenomas(15-10-7-6-5) and pituitary adenomas (24-18-24-14-11) in the female treatment groups. The incidences of the remaining tumours were not distributed in a treatment-related manner and comparable with those known from own historical control collectives. The number of cases, in which a tumour type occurred in one group only was comparable in the 3000/4000 ppm and 0 ppm group (for details please refer to Tables 16 in "any other information on results incl. tables".). The overall tumour incidence and the total number of benign and malignant neoplasms per group were not increased in treated males and females at all concentrations (for details please refer to Table 17 in "any other information on results incl. tables"). The number of malignant tumours was even reduced in males receiving 200 ppm and above. No remarkable differences were observed deaths in the number of tumour bearing rats or animals with a benign, malignant or benign and malignant tumour between the control and treatment groups for both, intercurrent or scheduled deaths (for details please refer to Table 18 in "any other information on results incl. tables"). As regards the time of occurrence of the tumours, there were no significant differences between treated rats and controls (for details please refer to Table 19 in "any other information on results incl. tables").

Other findings: Cholinesterase activities (ChE)
No significant ChE inhibition occurred in the plasma. The 24% lower plasma ChE activity of 3000 ppm males (Week 78) is not regarded as toxicologically relevant, because this was an isolated event, where in the control group relatively high activities were measured compared to those of the other time points. In addition, plasma ChE activity was even not affected when e.g. ChE activity in erythrocytes was inhibited by 90%. No remarkable ChE inhibition in erythrocytes were detected in males and females up to 200 ppm. At higher concentrations a dose-dependently and significantly lower ChE activity (inhibited up to 82% in males and 90% in females) was measured compared to control means. The significantly lower activity in 200 ppm females in Week 52/53 is considered to be incidental, because activity at this dose level was not remarkably inhibited at the other three time points, where stronger effects were noted in the two highest dose groups. Also in Week 52/53, a relatively high activity in controls may have contributed. Up to 3000 ppm (males) and 200 ppm (females) no significant or biologically relevant effect was noted on the brain ChE activity. In females there was a significantly reduced activity at 1000 and 4000 ppm (up to 41%). The inhibition values of 13 and 17% in 1000 ppm females and those of 14% calculated for 3000 ppm males at termination might be of questionable toxicological relevance.
Key result
Dose descriptor:
NOAEL
Remarks:
neoplastic
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no carcinogenic effects observed up to this dose level
Remarks on result:
other: corresponding to: 52.7 mg/kg bw/day (males) / 75.4 mg/kg bw/day (females)
Key result
Dose descriptor:
LOAEL
Remarks:
neoplastic
Effect level:
3 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: neoplastic
Remarks on result:
other: corresponding to: 170.4 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Remarks:
neoplastic
Effect level:
4 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: neoplastic
Remarks on result:
other: corresponding to: 326.7 mg/kg bw/day
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 ppm
System:
nervous system
Organ:
other: cholinesterase activity inhibited in erythrocytes (1000 ppm and above) and brain (4000 ppm) (statistically significant inhibition by 20% or more in erythrocytes or brain is considered a clear toxicological effect, please refer to chapter 7.5.1 1997f)
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Table 1: Cumulative Mortality data (number of dead animals up to week indicated)

Dose (ppm)

0

50

200

1000

3000

sex (male)

Weeks 0-52 (n = 60)

Week 26

0

1

0

0

0

Week 53

0

1

0

2

1

Weeks 53-104 (n = 50)

Week 79

5

4

1

5

1

Week 104

19

17

21

17

13

Dose (ppm)

0

50

200

1000

4000

sex (female)

Weeks 0-52 (n = 60)

Week 26

0

0

0

0

0

Week 53

0

2

1

0

2

Weeks 53-104 (n = 50)

Week 79

3

7

8

8

9

Week 104

13

19

22

20

21

 

Table 2: Body weights [g]

sex

m

m

m

m

m

f

f

f

f

f

Dose (ppm)

0

50

200

1000

3000

0

50

200

1000

4000

0

1

2

3

4

5

6

7

8

9

10

11

12

13

15

17

19

21

23

25

27

29

31

33

35

37

39

41

43

45

47

49

51

53

55

57

59

61

63

65

67

69

71

73

75

77

79

81

83

85

87

89

91

93

95

97

99

101

103

105

155

203

239

272

299

322

336

362

378

382

393

403

412

427

435

456

463

471

472

477

482

492

492

502

500

510

515

519

509

517

519

523

526

531

532

535

535

542

542

542

543

553

552

553

558

550

551

558

555

559

554

553

550

549

550

553

551

544

542

543

156

203

243

276

304

325

344

362

378

394

406

417

428

436

448

465

468

479

480

486

494

498

503

509

507

515

518

521

519

523

521

528

532

530

531

534

534

536

540

539

541

548

549

548

550

552

543

551

549

548

542

537

529

530

529

534

530

520

508 +

511

155

203

245

278

306

329

348

364

378

395

406

417

423

430

444

459

463

470

480

487

493

495

503

507

509

508

514

516

515

518

520

524

529

533

534

538

539

539

541

541

542

547

545

541

544

539

536

536

538

532

520 +

522

513 +

527

528

522

519

510 +

514

529

154

200

241

275

301

326

341

356

368

385

401

400

417

420

438

448

455

464

472

480

483

488

496

495

502

509

507

510

512

512

513

519

522

525

527

530

534

533

532

538

542

549

547

547

550

550

552

556

555

553

549

547

533

542

535

531

528

523

518

515

152

189++

238

264

289

312

330

344++

360++

372

382

391

401

401++

421

427++

441+

449+

454

463

466

468+

480

479+

485

483++

486++

489++

492

490++

490++

496++

500++

502++

505+

506++

513

511++

512++

515+

516+

521++

519++

516++

518++

512 ++

523 +

524 ++

527 +

526 +

518 +

513 ++

493 ++

504 ++

498 ++

497 ++

492 ++

495 ++

491 ++

483 ++

130

149

163

176

186

196

205

212

217

222

225

229

234

242

242

247

249

255

258

260

261

267

271

277

276

280

284

286

289

290

295

296

301

302

304

309

311

312

314

315

319

323

322

325

329

333

328

333

333

336

327

327

329

327

326

328

323

323

318

313

127

145

161

173

183

191

200

208

212

216

222

226

230

234

240

243

247

251

254

257

257

261

266

271

273

275

279

281

284

285

289

288

291

291

294

298

300

303

307

307

309

316

314

318

321

323

321

329

330

331

326

325

324

324

325

327

325

324

315

325

130

150

166

179

190

199

207

211

217

223

228

232

238

239

244

249

252

258

260

263

264

266

272

276

280

281

285

288

289

289

293

293

297

302

304

307

309

308

312

315

316

322

322

325

327

328

327

332

333

337

332

330

331

327

329

326

325

320

312

341

128

147

164

178

186

196

203

207

213

219

221

228

231

234

237

242

246

249

252

254

257

257

261

266

270

270

274

276

276+

275+

280+

279++

282++

284+

287+

291+

292+

294+

300

301

303

308

307

311

313

314

310

320

320

320

316

314

317

317

313

311

311

312

307

320

130

147

167

179

189

196

202

205

209

215

217

222

225

226++

230+

234++

234++

238++

242++

245++

246++

248++

252++

253++

257++

258++

262++

264++

264++

263++

266++

266++

268++

270++

271++

274++

275++

279++

278++

280++

281++

285++

283++

283++

286++

284 ++

284 ++

289 ++

291 ++

288 ++

286 ++

288 ++

287 ++

289 ++

289 ++

286 ++

283 ++

284 ++

281 ++

295

+ difference against control for p<0.05

++ difference against control for p<0.01

 

Table 3: Mean daily and cumulative intake of food and test compound

Intake food

 

 

 

g/animal

g/kg bw

Dose (ppm)

Sex

Days

Total

per Day

Total

per Day

0

m

708

14715

20.8

35813

50.6

50

m

708

14923

21.1

35653

50.4

200

m

708

15169

21.4

36331

51.3

1000

m

708

15391

21.7

37307

52.7

3000

m

708

15807

22.3

40218

56.8

0

f

708

12072

17.1

48515

68.5

50

f

708

12522

17.7

51527

72.8

200

f

708

12729

18.0

51524

72.8

1000

f

708

12712

18.0

53372

75.4

4000

f

708

13177

18.6

57821

81.7

Intake of test compound

 

 

 

mg/animal

mg/kg bw

Dose (ppm)

Sex

Days

Total

per Day

Total

per Day

50

m

708

746

1.1

1783

2.5

200

m

708

3034

4.3

7266

10.3

1000

m

708

15391

21.7

37307

52.7

3000

m

708

47421

67.0

120654

170.4

50

f

708

626

0.9

2576

3.6

200

f

708

2546

3.6

10305

14.6

1000

f

708

12712

18.0

53372

75.4

4000

f

708

52707

74.4

231285

326.7

 

Table 4: Mean daily cumulative water intake

Intake of water

 

 

 

g/animal

g/kg bw

Dose (ppm)

Sex

Days

Total

per Day

Total

per Day

0

m

708

18267

25.9

40827

57.8

50

m

708

18971

26.9

41396

58.6

200

m

708

19254

27.3

42187

59.8

1000

m

708

19034

27.0

42158

59.7

3000

m

708

19391

27.5

44915

63.6

0

f

708

18478

26.2

67633

96.8

50

f

708

18745

26.6

70078

99.3

200

f

708

18848

26.7

68707

97.3

1000

f

708

18539

26.3

70478

99.8

4000

f

708

17344

24.6

69761

98.8

 

Table 5: Ophthalmological findings at termination (Incidences in %)

sex

m

m

m

m

f

f

Dose (ppm)

0

200

1000

3000

0

4000

No. of eyes examined

61

71

61

73

76

58

Turbid cornea, including vascularisation of cornea

1.6

7.0

1.6

8.2

0

3.4

Turbid cornea, including vascularisation of cornea

Snow ball like turbidities between lens and vitreous

body (= posterior lenticular rupture)

11.4

4.2

8.2

13.7

7.9

0

Diffuse retrolenticular opacity (all degrees)

24.6

9.8

18.0

17.8

10.5

8.6

Wedge shaped waterclefts in the cortex, slight

18.0

7.0

8.2

8.2

3.9

5.1

Wedge shaped waterclefts in the cortex, moderate

0

0

3.3

1.4

0

1.7

Lens opacity in the whole cortex, slight

1.6

1.4

0

2.7

2.6

1.7

Lens opacity in the whole cortex, moderate

1.6

1.4

1.6

5.5

3.9

0

Lens opacity in the whole cortex, severe

1.6

0

6.5

5.5

6.6

3.4

 

Table 6: Hematology

Dose

 

 

LEU

ERY

HB

HCT

MCV

MCH

MCHC

RETI

HEINZ

THRO

HQUICK

ppm

Sex

Week

109/L

109/L

g/L

L/L

fl

pg

g/LERY

0/00

0/00

109/L

sec

0

m

26/27

10.3

9.04

147

0.459

50.8

16.3

320

18

0

1040

25.4

50

m

26/27

8.7

8.91

147

0.453

50.9

16.5

324

19

0

998

25.2

200

m

26/27

8.3

8.51

141

0.439

51.6

16.5

320

18

0

1109

24.7

1000

m

26/27

8.3

9.06

141

0.434

48.0++

15.6

325

17

0

1053

26.2

3000

m

26/27

8.0

8.58

138+

0.425++

49.6

16.1

325

17

1

982

25.4

0

m

52

9.5

9.42

153

0.487

51.8

16.2

313

16

0

1082

26.3

50

m

52

8.5

9.17

149

0.479

52.3

16.3

312

17

0

1059

26.0

200

m

52

8.4

8.87+

148

0.471

53.1

16.7

314

18

0

1172

26.1

1000

m

52

7.4

9.37

146

0.462

49.4

15.6

315

20

0

1082

25.4

3000

m

52

8.2

9.29

146

0.472

50.8

15.7

309

20

0

1026

26.3

0

m

78

8.0

9.04

151

0.476

52.7

16.8

318

17

0

1091

24.7

50

m

78

7.3

8.99

149

0.472

52.5

16.6

316

14

0

1073

24.6

200

m

78

6.7

8.20+

141

0.441

53.7

17.2

319

16

0

1277

24.0

1000

m

78

6.4

8.93

141

0.441

49.5+

15.9+

321

17

0

1041

24.1

3000

m

78

7.5

8.77

139

0.440

50.2

15.9+

316

15

0

998

24.1

0

m

103/104

9.2

9.26

160

0.512

55.4

17.3

312

20

0

1220

25.9

50

m

103/104

7.9

8.63

148

0.457+

52.9

17.1

322++

27

0

1158

25.0

200

m

103/104

7.9

8.85

154

0.473

53.5

17.5

326++

16

0

1156

24.2

1000

m

103/104

7.3

8.73

144

0.443++

50.9++

16.5

324++

23

0

1186

24.8

3000

m

103/104

6.9

9.07

150

0.466

51.5++

16.5

321+

29

0

1081

25.8

0

f

26/27

6.0

8.21

143

0.439

53.5

17.4

325

15

0

915

24.5

50

f

26/27

6.2

7.94

136

0.416

52.4

17.1

328

15

0

978

23.7

200

f

26/27

5.9

8.10

139

0.422

52.2

17.2

330

14

0

979

23.8

1000

f

26/27

6.2

8.01

137

0.424

52.8

17.1

325

17

0

955

23.8

4000

f

26/27

5.8

8.57

139

0.434

50.6++

16.2++

320

17

0

1046++

22.4

 

0

f

52

4.6

8.03

140

0.440

54.8

17.4

318

20

0

809

24.7

50

f

52

5.8

8.15

138

0.437

53.6

17.0

317

18

0

921

25.4

200

f

52

5.7

8.26

142

0.440

53.4

17.2

322

17

0

956

25.2

1000

f

52

5.7

8.25

140

0.447

54.2

17.0

314

19

0

897

24.1

4000

f

52

5.8

8.36

136

0.440

53.1

16.4+

309++

20

0

1017+

23.8

0

f

78

4.1

8.33

146

0.454

54.6

17.5

321

25

0

858

23.2

50

f

78

4.6

7.79

136

0.429

56.6

17.8

316

44

0

795

23.5

200

f

78

5.6

8.24

142

0.443

53.8

17.3

321

31

0

840

23.4

1000

f

78

4.3

8.16

143

0.451

55.4

17.6

317

31

0

848

26.2++

4000

f

78

4.7

8.06

133

0.431

53.6

16.6

309++

36

0

930

24.8+

0

f

103/104

5.6

8.29

151

0.453

54.9

18.3

333

31

0

1003

24.0

50

f

103/104

4.7

8.34

151

0.454

54.5

18.1

333

28

0

984

23.7

200

f

103/104

6.8

8.22

149

0.450

54.7

18.1

330

26

0

994

24.6

1000

f

103/104

4.3

8.33

151

0.463

55.7

18.1

325

29

0

948

24.8

4000

f

103/104

5.1

8.22

143  

0.443

 54.2 

17.4 

322+ 

 31 

0 

1066 

23.9 

+ difference against control for p<0.05

++ difference against control for p<0.01

 

Table 7: Differential blood count

Dose

Week

Sex

LYM

SEGM

EOS

MONO

BAND

ppm

 

 

%

%

%

%

%

0

26/27

m

87.5

8.0

0.2

4.3

0.0

50

26/27

m

89.8

6.5

0.3

3.4

0.0

200

26/27

m

86.0

10.1

0.6

3.3

0.0

1000

26/27

m

87.9

8.1

0.7

3.3

0.0

3000

26/27

m

89.4

7.6

0.6

2.5

0.0

0

52

m

79.7

12.7

1.0

6.6

0.0

50

52

m

78.8

11.7

1.8

7.7

0.0

200

52

m

79.1

12.7

0.9

7.3

0.0

1000

52

m

77.5

13.6

1.4

7.6

0.0

3000

52

m

78.0

13.7

0.6

7.8

0.0

0

78

m

78.4

13.9

1.5

6.2

0.0

50

78

m

75.9

16.2

2.2

5.6

0.0

200

78

m

74.9

17.4

1.7

6.2

0.0

1000

78

m

74.7

18.9

1.1

5.3

0.0

3000

78

m

77.6

17.0

0.8

4.6

0.0

0

103/104

m

67.8

23.5

1.2

7.4

0.0

50

103/104

m

62.7

29.7

1.7

6.0

0.0

200

103/104

m

70.0

23.6

1.5

4.8

0.1

1000

103/104

m

66.6

25.4

2.6

5.5

0.0

3000

103/104

m

71.8

23.0

1.2

4.2

0.0

0

26/27

f

88.4

8.4

0.8

2.5

0.0

50

26/27

f

86.0

11.3

0.8

1.9

0.0

200

26/27

f

89.5

7.8

0.8

1.9

0.0

1000

26/27

f

90.7

8.2

0.4

0.7+

0.0

4000

26/27

f

88.7

8.5

0.8

2.0

0.0

0

52

f

79.9

14.0

1.4

4.7

0.0

50

52

f

76.5

16.5

1.0

6.0

0.0

200

52

f

80.7

13.3

1.2

4.8

0.0

1000

52

f

80.5

15.0

0.6

3.9

0.0

4000

52

f

86.4

9.7

0.5

3.4

0.0

0

78

f

71.8

23.7

1.2

3.3

0.0

50

78

f

75.7

16.2

1.7

6.4

0.1

200

78

f

65.9

26.9

1.0

6.3

0.0

1000

78

f

73.5

19.8

1.2

5.6

0.0

4000

78

f

78.4

16.0

1.1

4.5

0.0

0

103/104

f

70.4

27.4

1.2

1.1

0.0

50

103/104

f

69.3

28.8

0.8

1.1

0.0

200

103/104

f

60.4

37.3

1.3

1.0

0.0

1000

103/104

f

75.8

22.0

1.0

1.2

0.0

4000

 103/104

f

73.1

 23.8

 1.6

 1.6

0.0

+ difference against control for p<0.05

++ difference against control for p<0.01

 

Table 8: Clinical Chemistry

Dose

Week

Sex

ASAT (GOT)

ALAT (GPT)

APh

GGT

GLDH

ppm

 

 

U/L

U/L

U/L

U/L

U/L

0

26/27

m

41.3

38.9

190

1

5.7

50

26/27

m

38.8

40.1

186

1

5.4

200

26/27

m

44.4

40.3

171

2

7.9

1000

26/27

m

38.5

40.4

157+

2

5.0

3000

26/27

m

35.1

37.7

150+

3++

3.8

0

52

m

39.9

42.2

184

1

16.2

50

52

m

40.5

44.l

193

1

22.8

200

52

m

36.0

37.9

162

2

12.0

1000

52

m

31.2

37.4

154+

1

12.6

3000

52

m

34.6

41.0

156+

2

12.8

0

78

m

38.8

36.5

157

1

16.0

50

78

m

42.5

43.8

178

2

22.0

200

78

m

35.1

32.1

142

1

14.0

1000

78

m

33.2

36.0

141

1

17.9

3000

78

m

32.7

35.7

144

2

20.0

0

103/104

m

40.2

37.5

164

3

21.4

50

103/104

m

42.2

33.1

175

2

21.6

200

103/104

m

37.4

32.3

134

2

10.8

1000

103/104

m

37.4

29.8

137

3

14.8

3000

103/104

m

36.9

35.3

140

3

19.0

0

26/27

f

40.7

43.4

121

0

11.5

50

26/27

f

48.0

43.6

133

0

18.7

200

26/27

f

41.0

44.9

122

0

11.3

1000

26/27

f

32.3+

41.9

103

0

6.8

4000

26/27

f

30.9++

42.6

105

1

1.0

0

52

f

44.7

42.6

120

0

17.2

50

52

f

46.8

44.1

134

0

22.9

200

52

f

42.5

43.8

111

0

20.4

1000

52

f

55.4

55.2

99

0

63.3

4000

52

f

33.3

45.0

118

0

9.2

0

78

f

59.4

45.7

100

1

59.2

50

78

f

89.4

62.2

113

0

104.8

200

78

f

73.9

54.4

91

0

87.7

1000

78

f

40.3

36.3

81+

1

29.1

4000

78

f

41.2

37.5

90

2+

18.4

0

103/104

f

61.3

45.9

114

0

35.5

50

103/104

f

61.3

49.0

114

1

53.7

200

103/104

f

76.6

53.8

102

0

70.9

1000

103/104

f

45.7

41.2

92

0

26.4

4000

103/104

f

 43.4

 43.6 

102 

2++ 

41.5 

+ difference against control for p<0.05

++ difference against control for p<0.01

 

Table 9: Activities and inhibition of cholinesterases (CHE)

Dose

Week

Sex

CHE/Brain

CHE/Plasma

CHE/Erythrocytes

ppm

 

 

U/g

% Inhibition

kU/L

% Inhibition

kU/L

% Inhibition

0

27

m

-

 

0.50

 

1.06

 

50

27

m

-

 

0.52

 

1.06

 

200

27

m

-

 

0.52

 

0.88

17

1000

27

m

-

 

0.50

 

0.65+

39

3000

27

m

-

 

0.45

10

0.32++

70

0

52/53

m

10.64

 

0.62

 

1.14

 

50

52/53

m

10.46

2

0.68

 

1.15

 

200

52/53

m

10.56

 

0.63

 

1.01

11

1000

52/53

m

10.26

4

0.61

2

1.00

12

3000

52/53

m

9.54

10

0.56

10

0.57++

50

0

78

m

-

 

0.72

 

1.24

 

50

78

m

-

 

0.71

 

1.35

 

200

78

m

-

 

0.71

1

1.16

6

1000

78

m

-

 

0.63

13

0.84+

32

3000

78

m

 

 

0.55

24

0.55++

55

0

103/104/105

m

9.88

 

0.66

 

0.83

 

50

103/104/105

m

9.87

 

0.95

 

0.76

8

200

103/104/105

m

9.86

 

0.86

 

0.70

16

1000

103/104/105

m

9.47

4

0.65

2

0.50++

40

3000

103/104/105

m

8.50

14

0.55

17

0.15++

82

0

26/27

f

-

 

2.47

 

0.77

 

50

26/27

f

-

 

2.24

9

0.83

 

200

26/27

f

-

 

2.15

13

0.70

9

1000

26/27

f

-

 

2.57

 

0.38++

51

4000

26/27

f

-

 

2.30

7

0.08++

90

0

52/53

f

11.28

 

2.39

 

1.24

 

50

52/53

f

10.82+

4

2.21

8

1.17

6

200

52/53

f

10.80

4

2.39

 

0.96+

23

1000

52/53

f

9.41++

17

2.73

 

0.58++

53

4000

52/53

f

7.07++

37

2.49

 

0.34++

73

0

78

f

-

 

2.18

 

1.19

 

50

78

f

-

 

2.03

7

1.18

1

200

78

f

-

 

2.12

3

1.01

15

1000

78

f

-

 

2.45

 

0.45++

62

4000

78

f

-

 

2.22

 

0.13++

89

0

103/104/105

f

10.22

 

2.25

 

0.74

 

50

103/104/105

f

10.02

2

2.31

 

0.66

11

200

103/104/105

f

9.86

4

1.95

13

0.73

1

1000

103/104/105

f

8.91++

13

2.11

6

0.28++

62

 4000

103/104/105

f

6.02++

41

 2.08

 8

0.08++

89

+ difference against control for p<0.05

++ difference against control for p<0.01

 

Table 10: Determination of urine pH week 98/99

 

pH mean value

Sex

male

female

Dose (ppm)

0

7.55

7.20

50

7.57

7.47

200

7.32

7.12

1000

7.45

7.64

3000

7.34

-

4000

-

7.67

 

Table 11: Urinalyses

Dose (ppm)

Week

Sex

VOL (ml)

Density (g/L)

PROT*Vol (mg)

PROT (g/L)

0

26

m

13.9

1023

20.3

1.78

50

26

m

14.8

1021

30.1

2.88

200

26

m

12.2

1025

56.5

5.23

1000

26

m

10.8

1032

26.7

3.29

3000

26

m

15.6

1020

31.1

3.07

0

52

m

6.3

1036

33.5

5.73

50

52

m

8.2

1035

57.9

7.95

200

52

m

7.0

1039

101.2

13.71

1000

52

m

11.2

1032

61.4

9.03

3000

52

m

9.3

1034

58.2

7.84

0

79

m

7.1

1037

35.2

5.35

50

79

m

7.8

1030

42.9

5.49

200

79

m

12.3

1023

76.1

6.53

1000

79

m

7.5

1025

36.4

5.99

3000

79

m

8.4

1028

43.5

6.37

0

103

m

11.6

1024

75.9

7.70

50

103

m

8.6

1031

77.2

8.98

200

103

m

10.9

1024

84.1

8.65

1000

103

m

8.8

1030

91.4

9.67

3000

103

m

11.8

1024

75.7

6.64

0

26

f

8.5

1026

5.5

0.68

50

26

f

14.0

1016

2.8

0.25

200

26

f

12.5

1022

2.4

0.24+

1000

26

f

6.9

1028

1.8

0.31

4000

26

f

7.3

1026

1.9

0.34

0

52

f

8.4

1029

13.3

1.25

50

52

f

9.0

1021

3.2

0.37

200

52

f

10.7

1022

4.5

0.45

1000

52

f

12.5

1015+

2.5

0.32+

4000

52

f

8.4

1030

1.8

0.29+

0

79

f

5.8

1026

10.1

1.85

50

79

f

8.0

1020+

6.3

0.91

200

79

f

11.2+

1019+

10.9

1.25

1000

79

f

10.8+

1015+

6.0

0.66

4000

79

f

10.6

1018+

3.3

0.42

0

103

f

8.4

1024

46.0

4.94

50

103

f

10.0

1021

42.5

4.80

200

103

f

7.9

1024

23.6

2.85

1000

103

f

9.8

1022

19.7

1.16+

4000 

103

 f 

7.9

1029

7.5

1.14+

+ difference against control for p<0.05

++ difference against control for p<0.01

 

Table 12: Absolute and relative organ weights (mg/100 g bw) – interim kill

Dose

Sex

Body weight

Brain

Adrenals

Heart

Liver

Spleen

Kidneys

Testes

Ovaries

Absolute organ weights

g

mg

mg

mg

mg

mg

mg

mg

mg

0

m

517

2052

54

1402

17938

853

2910

3844

-

50

m

557

2159+

53

1534

19013

944

3220

4062

-

200

m

528

2064

54

1467

17702

881

3085

3815

-

1000

m

531

2128

52

1560

19351

801

3054

3652

-

3000

m

519

2105

56

1439

21255+

715

2998

4051

-

0

f

312

1912

84

1023

10016

535

1994

-

172

50

f

302

1890

83

1031

9690

561

2057

-

173

200

f

296

1920

83

1059

9442

476

2061

-

197

1000

f

285

1879

80

988

10299

684

1983

-

204

4000

f

261++

1827

69

974

11901++

488

1856

-

162

Relative organ weights

Body weight

Brain

Adrenals

Heart

Liver

Spleen

Kidneys

Testes

Ovaries

g

mg

mg

mg

mg

mg

mg

mg

mg

0

m

517

400

10

272

3480

166

561

749

-

50

m

557

390

10

276

3424

170

580

731

-

200

m

528

393

10

279

3347

167

585

723

-

1000

m

531

403

10

296

3649

151

579

697

-

3000

m

519

406

11

278

4094+

137

578

782

-

0

f

312

620

27

331

3235

173

642

-

55

50

f

302

628

28

342

3205

186

681

-

57

200

f

296

652

28

357

3196

195

696

-

68

1000

f

285

660

28

347

3615+

235

696

-

71

4000

f

261++

704++

27

375+

4584++

186

711+

-

63

+ difference against control for p<0.05

++ difference against control for p<0.01

 

Table 13: Absolute and relative organ weights (mg/100 g bw) – terminal kill

Dose

Sex

Body weight

Brain

Adrenals

Heart

Liver

Spleen

Kidneys

Testes

Ovaries

Absolute organ weights

g

mg

mg

mg

mg

mg

mg

mg

mg

0

m

534

2176

64

1866

18013

1217

3491

3785

-

50

m

507

2191

68

1960

17363

1098

3429

3929

-

200

m

508

2172

58

1830

18083

1157

3611

3847

-

1000

m

513

2165

71

18368

18368

1035+

3407

4048

-

3000

m

483++

2118

78

19494

19494

937++

3369

4029

-

0

f

318

1931

84

11395

11395

721

2295

-

216

50

f

318

1957

77

11784

11784

714

2380

-

173

200

f

312

1947

82

11347

11347

738

2328

-

181

1000

f

308

1938

69

11579

11579

671

2197

-

193

4000

f

282++

1914

71

13476++

13476++

585++

2190

-

194

Relative organ weights

Body weight

Brain

Adrenals

Heart

Liver

Spleen

Kidneys

Testes

Ovaries

g

mg

mg

mg

mg

mg

mg

mg

mg

0

m

534

410

12

351

3369

229

655

712

-

50

m

507

435

14

387

3416

229

680

776

-

200

m

508

434

12

367

3608

230

721

756

-

1000

m

513

426

14

364

3608

203

669

793

-

3000

m

483++

483++

17

400+

4050++

196+

706

835++

-

0

f

318

617

27

448

3608

229

728

-

68

50

f

318

625

25

444

3758

229

754

-

54

200

f

312

633

27

440

3674

239

754

-

58

1000

f

308

638

23

437

3773

219

716

-

62

4000

f

282++

682++

25

470

4795++

208

781

-

69

+ difference against control for p<0.05

++ difference against control for p<0.01

 

Table 14: Number of rats with remarkable non-neoplastic lesions – interim kill

Sex

 

m

m

m

m

 

f

f

f

f

f

Dose

(ppm)

0

50

200

1000

3000

0

50

200

1000

4000

Organ/Finding

Number Animals

10

10

10

10

10

10

10

10

10

10

Adrenal Glands

- increased vacuolation in zona fascicu.*

§4

6

4

6

10

§2

0

0

0

9

Liver

- hepatocellular hypertrophy (centilobul.)

§0

0

0

6

10

§0

0

0

3

10

Urinary Bladder

- transitional hyperplasia

#0

0

2

2

3

§0

0

0

0

3

*= increased cytoplasmic vacuolisation in zona fasciculata

# trend (p < 0.05)

§ trend (p < 0.01)

 

Table 15:Number of rats with remarkable non-neoplastic lesions – terminal kill

Sex

 

 

m

m

m

m

 

 

f

f

f

f

f

Dose

(ppm)

 

0

50

200

1000

3000

 

0

50

200

1000

4000

Organ/Finding

Number Animals

 

50

50

50

50

50

 

50

50

50

50

50

Liver

- eosinophilic foci

#

3

3

1

2

10

#

 

 

3

3

3

- focal degenerative changes (periportal)

§

 

 

 

2

13

 

 

 

 

 

1

- clear cell foci

 

36

34

31

35

28

 

6

9

6

9

1

- cytoplasmic changes (periportal)

§

 

 

 

2

22

 

 

 

 

 

 

- cytoplasmic changes (centrilobular)

§

 

 

1

 

14

§

1

3

3

8

38

- hepatocellular hypertrophy (centilobul.)

 

1

3

 

1

4

#

 

 

1

3

40

- peliotic foci

 

 

 

 

 

 

§

 

 

 

 

3

- single cell necrosis

 

 

 

 

 

 

§

4

7

5

5

16

Urinary Bladder

- transitional hyperplasia (focal)

§

 

 

 

 

4

 

1

 

 

 

3

- simple transitional hyperplasia (diffuse)

§

 

 

3

 

11

§

 

 

 

 

20

Skeletal Muscle

- atrophy

#

7

2

9

10

11

§

 

 

1

2

9

- fibre degeneration (thigh)

 

4

5

6

3

6

#

2

 

1

 

7

Sciatic Nerve

- degenerative myelinopathy

§

21

25

27

32

43

§

17

20

13

23

38

Thyroid Gland

- follicular hyperplasia

§

1

1

2

3

7

 

2

 

 

2

1

- mineralization of follicular colloid

#

33

28

31

35

39

§

13

13

17

17

43

Adrenal Glands

- increased vacuolation in zona fascicu.*

§

5

7

7

3

20

 

1

2

2

3

4

- degenerative changes in the cortex

 

5

3

3

3

3

 

19

14

11

8

4

Pituitary Gland

(pars distalis)

- hyperplasia (focal)

 

10

6

4

13

10

 

18

13

9

11

9

Stomach

(glandular mucosa)

- dilated glands

 

6

4

8

7

6

#

7

7

6

8

16

Tongue

- necrotizing arteritis

 

7

12

7

11

9

 

7

7

4

2

0

Heart

- cardiomyopathy

 

43

45

48

44

48

 

37

37

33

28

22

Kidneys

- chronic progressive nephropathy

 

47

47

48

44

48

 

41

32

36

26

21

- diffuse urothelial hyperplasia

 

10

18

16

8

15

 

35

35

30

21

19

Spleen

- pigment storage

 

19

24

31

20

27

§

9

9

11

15

23

# trend (p < 0.05)

§ trend (p < 0.01)

 

Table 16: Tumor incidences – Number with neoplastic lesions – terminal kill 

Sex

 

Males

Females

Dose

(ppm)

0

50

200

1000

3000

0

50

200

1000

4000

Organ/Findings

No.animals

50

50

50

50

50

50

50

50

50

50

 

Liver

No. exam.

50

 

49

 

50

 

50

 

50

 

50

 

49

 

50

 

50

 

50

- Hepatocellular Adenoma

 

1

 

1

 

 

 

1

 

 

1

- Hcpatocellular Adcnocarcinoma

 

1

 

 

 

 

 

 

 

 

 

-Cholangioma

 

 

 

 

 

 

 

 

 

1

 

- Cholangiocarcinoma

 

 

1

 

 

 

 

 

 

 

 

Heart

No. exam.

50

49

50

50

50

50

49

50

50

50

- Endocardial Tumor (mal.)

 

2

1

 

 

 

 

1

 

 

 

Kidneys

No. exam.

50

49

50

50

50

50

49

50

50

50

-Adenoma

 

 

 

 

1

 

 

 

 

 

 

- Lipoma

 

 

 

 

 

1

 

 

 

 

 

- Liposarcoma

 

 

 

1

 

 

 

 

 

 

 

Urinary Bladder

No. exam.

50

48

50

50

50

50

49

48

49

49

- Transitional Cell Papilloma

 

 

 

 

 

 

§

 

 

 

2

- Transitional Cell Carcinoma

 

 

 

 

 

 

 

 

 

 

1

Urethra (residual)&)

No. exam.

 

 

 

 

1

 

 

 

 

 

- Transitional Cell Carcinoma

 

 

 

 

 

1

 

 

 

 

 

LungsNo. Exam.

No. exam.

50

49

50

50

50

50

49

50

50

50

-  AlvcolarffironchiolarAdenoma

 

 

1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

-  Alveolar/UronchiolarCarcinoma

 

 

 

1

1

 

 

 

 

 

 

 

Metastasis primary siteunknown

 

 

1

 

 

 

 

 

1

 

 

Stomach

No. Exam.

No. exam.

50

49

50

50

50

50

49

50

50

50

- Forestomach Papilloma

 

 

 

 

 

 

 

 

 

 

1

Colon

No. exam.

50

48

50

50

50

50

49

49

49

49

- Fibrosarcoma

 

 

 

 

 

 

 

1

 

 

 

Intestine (residual)&)

No. exam.

 

 

 

 

 

 

 

1

2

2

-Fibroma

 

 

 

 

 

 

 

 

 

1

 

Pancreas

No. exam.

48

49

50

50

50

50

49

50

50

50

- Islet Cell Adenoma

 

1

1

2

3

 

1

 

 

 

 

- Acinar Cell Adenoma

 

 

 

 

 

1

 

 

 

1

 

Thymus

No. exam.

49

49

50

50

50

48

49

50

50

48

- Thymoma (benigne)

 

1

1

 

1

 

 

I

2

 

1

- Thymoma (maligne)

 

1

 

 

 

 

 

 

 

 

 

Mesent. Lymph Node

Mesent. Lymph NodeNo. Exam.

No. exam.

49

449

49

5049

50

50

50

50

50

50

50

50

49

49

50

50

49

49

50

50

- Hemangioma

 

1

1

1

 

1

 

 

 

 

 

Spleen

No. exam.

50

49

50

50

50

50

49

50

49

50

- Hemangiosarcoma

 

1

 

 

 

 

 

 

 

 

 

Skin/Other&)

No. exam.

9

3

5

5

4

6

2

2

2

2

- Schwannoma (mal.)

 

 

1

 

 

 

 

 

 

 

 

- Fibroma

 

 

 

 

1

 

 

 

 

 

 

- Fibrosarcoma

 

1

 

 

 

 

 

1

 

 

 

- Squamous Cell Carcinoma

 

2

1

 

1

 

 

 

 

 

 

- Keratoacanthoma

 

3

 

2

 

1

 

 

 

 

 

- Papilloma

 

 

 

 

 

1

 

 

 

 

 

- Hair Follicle Tumor (ben.)

 

 

 

1

 

 

 

 

 

 

 

-Lipoma

 

 

 

 

 

 

1

 

 

 

 

- Basal Cell Carcinoma

 

 

 

 

 

 

 

 

1

 

 

Mammary Gland/Region

No. exam.

-

-

-

-

-

50

49

50

50

50

-Adenoma

 

 

 

 

 

 

 

1

 

 

 

- Adenocarcinoma

 

 

 

 

 

 

3

1

6

 

2

- Adenocarcinoma arising in a fibroadenoma

 

 

 

 

 

 

1

 

 

 

1

- Fihroadenoma

 

 

 

 

 

 

15

10

7

6

5

- Tumor Mixed (mal.)

 

 

 

 

 

 

1

 

 

1

1

Brain

No. exam.

50

49

50

50

50

50

49

50

50

50

- Astrocytoma (ben.)

 

1

 

 

 

 

 

 

 

 

 

- Granular Cell Tumor (ben.)

 

 

 

 

 

1

 

1

 

 

 

Testes

No. exam.

50

49

50

50

50

-

-

-

-

-

- Leydig Cell Adenoma

 

1

4

 

1

2

 

 

 

 

 

Prostate

No. exam.

50

49

50

50

50

-

-

-

-

-

- Fibrosarcoma

 

 

 

 

1

 

 

 

 

 

 

Ovaries

No. exam.

-

-

-

-

-

50

49

50

50

50

- Granulosa Cell Tumor (ben.)

 

 

 

 

 

 

1

 

 

 

 

- Granulosa Cell Tumor (mal.)

 

 

 

 

 

 

 

 

 

 

1

- Tumor/Sex Cord (ben.)

 

 

 

 

 

 

 

1

1

1

 

Uterus

Uterus

No. exam.

No. Exam.

 

 

 

 

 

50

49

550

50

49

50

50

50

50

-Adenoma

 

 

 

 

 

 

 

 

 

1

 

- Adenocarcinoma

 

 

 

 

 

 

§ 2

2

3

3

8

- Adenosquamous Carcinoma

 

 

 

 

 

 

1

1

1

 

 

- Squamous Cell Carcinoma

 

 

 

 

 

 

 

 

 

 

1

- Schwannoma (mal.)

 

 

 

 

 

 

1

 

1

 

 

- Stromal Sarcoma

 

 

 

 

 

 

 

1

 

1

1

- Stromal Polyp

 

 

 

 

 

 

7

7

4

5

6

- Glandular Polyp

 

 

 

 

 

 

1

 

3

 

 

Vagina

No. exam.

-

-

-

-

-

50

49

50

50

50

- Granular Cell Tumor (ben.)

 

 

 

 

 

 

 

 

 

 

1

Adrenal Glands

No. exam.

50

49

50

50

50

50

49

50

50

50

- Cortical Adenoma

 

 

 

1

 

1

 

1

 

 

 

- Cortical Adenocarcinoma

 

 

 

 

 

 

1

 

 

 

 

- Medullary Tumor (ben.)

 

10

9

5

8

10

1

3

1

1

 

- Medullary Tumor (mal.)

 

2

1

1

1

 

 

 

1

 

 

Pituitary Gland

No. exam.

49

49

50

50

50

50

49

50

50

50

- Adenoma (Pars Distalis)

 

5

6

11

5

8

24

18

24

14

11

- Adenoma (Pars Intermedia)

 

 

 

 

 

 

1

 

 

 

1

- Adenocarcinoma (Pars Distalis)

 

 

 

 

 

 

1

 

1

 

 

Thyroid Gland

No. exam.

50

49

50

50

50

50

49

50

50

50

- C-Cell Adenoma

 

3

7

2

2

3

3

2

3

4

3

- C-Cell Adenocarcinoma

 

1

 

 

 

 

 

 

 

 

 

- Follicular Adenoma

 

#

 

 

1

2

1

1

1

1

1

- Follicular Adenocarcinoma

 

 

 

 

 

1

 

 

 

 

 

Bone/NOS&)

 

 

 

 

 

 

 

 

1

1

 

- Osteosarcoma

 

 

 

 

 

 

 

 

1

 

 

- Schwannoma (maligne)

 

 

 

 

 

 

 

 

 

1

 

Systemic Tumors

No. exam. 

50

49

50

50

50

50

49

50

50

50

- Lymphoma (maligne)

 

2

1

 

2

 

 

 

1

1

 

- Myeloid Leukemia

 

 

1

 

1

 

 

 

 

 

 

- Histiocytic Sarcoma

 

2

1

 

1

1

 

 

 

 

 

* in case of two tumors in a paired organ one tumor was taken into consideration

&) only investigated in case of macro lesions

# positive trend p<0.05

§ positive trend p<0.01

NOS = not otherwise specified

no. = number

exam. = examinations

 

 

Table 17: Number of Animals with neoplastic lesions – terminal kill

Sex

 

male

female

Dose ppm

 

0

50

200

1000

3000

0

50

200

1000

4000

Organ/Finding

No. animals

50

50

50

50

50

50

50

50

50

50

 

Body cavities &)

No. exam.

 

5

 

3

 

6

 

6

 

3

 

3

 

3

 

3

 

4

 

6

- Mesothelioma (benign)

 

1

 

 

 

 

 

 

 

 

 

- Lipoma

 

 

 

1

 

 

1

 

 

1

 

- Fibroma

 

 

 

 

 

 

1

 

 

 

 

Clitorial glands &)

No. exam.

 

 

 

 

 

 

1

1

 

 

- Squamous cell carcinoma

 

 

 

 

 

 

 

 

1

 

 

Number of benign tumors

 

29

29

27

23

32

58

47

47

36

33

Malignant tumors

 

15

10

3

7

3

11

8

18

7

16

Total tumors

 

44

39

30

30

35

69

55

65

43

49

* in case of two tumors in a paired organ one tumor was taken into consideration

&) only investigated in case of macro lesions

# positive trend p<0.05

§ positive trend p<0.01

NOS = not otherwise specified

no. = number

exam. = examinations

 

 

 

Table 18: Animals with tumors – terminal kill

Sex

Males

Females

Dose ppm

0

50

200

1000

3000

0

50

200

1000

4000

 

 

lntercurrent Deaths

No. of animals examined

21

19

21

17

13

12

20

24

20

22

Animals with tumors

12

11

13

7

5

10

13

19

12

15

Animals with benign tumors only

5

7

10

3

4

8

7

9

7

5

Animals with malignant tumors only

5

5

3

2

 

1

4

5

2

3

Animals with benign and malignant tumors

2

 

 

2

1

1

2

5

3

7

 

Terminal Kill

No. of animals examined

29

31

29

33

37

38

30

26

30

28

Animals with tumors

17

16

13

16

21

31

20

22

16

15

Animals with benign tumors only

10

11

13

13

19

25

19

15

14

10

Animals with malignant tumors only

2

2

 

2

1

1

 

 

 

1

Animals with benign and malignant tumors

5

3

 

1

1

5

1

7

2

4

 

All Animals

No. of animals examined

50

50

50

50

50

50

50

50

50

50

Animals with tumors

29

27

26

23

26

41

33

41

28

30

Animals with benign tumors only

15

18

23

16

23

33

26

24

21

15

Animals with malignant tumors only

7

7

3

4

1

2

4

5

2

4

Animals with benign and malignant tumors

7

3

 

3

2

6

3

12

5

11

 

 

Table 19: Occurrence of tumor bearing animals in time 

Sex

 

Males

Females

Dose ppm

 

0

50

200

1000

3000

0

50

200

1000

4000

Number of Animals with Neoplasms

 

 

 

 

 

 

 

 

 

 

 

Dying Prematurely

 

12

11

13

7

5

10

13

19

12

15

Neoplasms

benign

11

9

13

7

7

13

11

17

11

14

 

maligne

7

5

3

4

1

3

7

9

5

11

 

total

18

14

16

11

8

16

18

26

16

25

Number of Animals with Tumors

Week

0-13

 

 

1

 

 

 

 

 

 

 

 

 

13-26

 

 

 

 

 

 

 

 

 

 

 

 

27-39

 

 

 

 

 

 

 

 

 

 

 

 

40-52

 

 

 

 

1

 

 

1

1

 

 

 

53-65

 

1

2

 

 

 

 

 

 

2

1

 

66-78

 

1

 

1

1

 

1

2

4

1

2

 

79-91

 

4

2

6

2

2

3

3

8

4

7

 

92-Necropsy

 

6

7

6

3

3

6

7

6

5

5

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:
adopted 12th May 1981
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-1,2,3,4-tetrazole-1-carboxamide
EC Number:
605-140-1
Cas Number:
158237-07-1
Molecular formula:
C16H20ClN5O2
IUPAC Name:
4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-1,2,3,4-tetrazole-1-carboxamide

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Hsd/WIN: WU
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 5 - 6 weeks
- Weight at study initiation: 110 - 182 g (males), 102 - 158 g (females)
- Housing: 5 animals separated by sex in Type III Makrolon® and Ha cages. The cages containing the experimental animals were separated by groups and placed on shelves in order of ascending animal number. The position of the shelves was changed on a random basis every 4 weeks.
- Diet: Altromin® 1321 meal (Altromin GmbH, Lage) containing 1% peanut oil, ad libitum except during the urine collection period
- Water: tap water, ad libitum
- Acclimation period: 1 week

DETAILS OF FOOD AND WATER QUALITY:
The tap water complied with drinking water standards in accordance with the Deutsche Trinkwasserverordnung.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 55% ± 5%
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- The test substance was blended (using a mixing granulator manufactured by Loedige, Paderborn) with Altromin® 1321 containing 1% peanut oil to minimize dust formation (including 0 ppm concentration).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical investigations to demonstrate homogeneity and stability of the test compound in diet preparations were done prior to commencement of the study. The test substance content of diet mixtures fed to the animals was checked analytically at regular intervals throughout the study (start of study, randomly each 3 month period, end of study). Per dose one sample of the food mixtures was taken on the day the mixture was prepared, and another was taken after being kept underanimal room conditions for the feeding period (7 days). All these samples were kept deep frozen (at temperatures of approx. -20°C) until analysis.
Duration of treatment / exposure:
24 months
Frequency of treatment:
daily per feed, ad libitum
Doses / concentrationsopen allclose all
Dose / conc.:
50 ppm
Remarks:
corresponding to
2.5 mg/kg bw/day (males)
3.4 mg/kg bw/day (females)
Dose / conc.:
200 ppm
Remarks:
corresponding to
10.3 mg/kg bw/day (males)
14.6 mg/kg bw/day (females)
Dose / conc.:
1 000 ppm
Remarks:
corresponding to
52.7 mg/kg bw/day (males)
75.4 mg/kg bw/day (females)
Dose / conc.:
3 000 ppm
Remarks:
corresponding to
170.4 mg/kg bw/day (males)
Dose / conc.:
4 000 ppm
Remarks:
corresponding to
326.7 mg/kg bw/day (females)
No. of animals per sex per dose:
60
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dosages were selected on the basis of the results of a subchronic feeding study in Wistar rats in which the test substance had been administered to 10 males and 10 females at doses of 0, 20, 100, 400, 1600 and 6400 ppm for 13 weeks. In this study concentrations of up to 400 ppm were tolerated without adverse effects. At higher concentrations body weight depression, effects on blood parameters (essentially reduced hemoglobin and hematocrit values and elevated reticulocyte means), inhibition of the cholinesterase activity (erythrocytes) and signs of changed liver function such as altered biochemical parameters (enhanced cholesterol plasma levels, reduced contents of triglycerides and unesterified fatty acids, lower albumin means), enzyme induction, increased liver weights and occurrence of hepatocellular hypertrophy were evident. Rats receiving 6400 ppm exhibited diffuse hyperplasia of the bladder urothelium as well. In further electron-microscopical investigations on the urinary bladder cytotoxicity with mild regenerative hyperplasia was detected in 6400 ppm males (females were not investigated) with higher frequency than in controls

- Rationale for animal assignment: The rats were weighed individually beforehand and the required number of animals were then grouped by weight (light, midde and heavy subgroups) and randomly distributed into large containers.
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily and daily at weekends and on bank holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
Parameters: orifices, posture, general behavior, respiration and excretory products were carefully examined, with any significant findings were registered. Animals that became ill or had developed neoplasms that could lead to death were kept apart, observed more frequently, and killed prematurely if death was imminent.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly from start to Week 13, and every 2 weeks thereafter up to Week 105

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: weekly within the first 13 weeks of treatment and every 4 weeks thereafter up to Week 101

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations and dose groups that were examined: during the first two weeks of the study in all rats. In Week 54 and prior to the final bleeding all living animals scheduled for the final necropsy from the control group and the 3000 or 4000 ppm dose groups were examined. At study termination 200 and 1000 ppm males were investigated, additionally.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Weeks 26/27, 52, 78 and 105/106
- Anaesthetic used for blood collection: Yes, for glucose and deproteinized whole blood only
- Animals fasted: Yes
- How many animals: 10
- Parameters analysed: differential blood count, erythrocyt morphology, erythrocyte count, Heinz bodies, hemoglobin, hematocrit, leucocyte count, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, platelet count, thromboplastin time, reticulocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Weeks 26/27, 52, 78 and 105/106
- Animals fasted: Yes
- How many animals: 10
- Parameters analysed: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, glutamate dehydrogenase, glucose, bilirubin, albumin, cholesterol, creatinine, total protein, triglyceride, urea, calcium, sodium, potassium, chloride and inorganic phosphate

URINALYSIS: Yes
- Time schedule for collection of urine: Weeks 26/27, 52, 79 and 98/99
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters analysed: sediment, bilirubin, blood, glucose, ketone bodies, pH, protein, urobilinogen (semiquantitative), density, volume, total protein (quantitative)

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER:
Cholinesterase activity in brain
- Time schedule: Weeks 52/53 and 104/105

Cholinesterase activities in plasma and erythrocytes
- Time schedule: Week 26/27, measurement were repeated for males in the following week due to some implausible values

Determination of crystals in urine sediment
- Time schedule: Week 103
- Dose groups that were examined: 8 animals, in controls and rats of the 3000 and 4000 ppm groups
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Organ weights: brain, heart, liver, spleen, kidneys, adrenals, testes, ovaries

HISTOPATHOLOGY: Yes, the following organs and tissues were fixed for analysis: Adrenals , Aorta , Brain (3 locations) [in some cases half brain] , Caecum, Colon, Duodenum, Epididymides , Esophagus, Eyes (with eyelids), Exorbital lacrimal glands, Femur (incl. bone marrow and knee joint), Harderian glands, Head-Nose-Pharynx area, Heart, lleum, Jejunum, Kidneys, Larynx, Liver, Lungs, Lymph nodes (mandibular and mesenteric), Mammary glands, Optic nerve, Ovaries (incl. oviduct), Pancreas, Pituitary, Prostate, Rectum, Residual Intestine, Salivary glands, Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (3 regions), Spleen, Sternum (with bone marrow), Stomach (with forestomach), Tattooed ears, Testes, Thymus (if present), Thyroid (parathyroid), Tongue, Trachea, Ureters, Urethra, Urinary bladder, Uterus, Vagina, Zymbal glands and all tissues showing abnormalities
Statistics:
Dunnett-Test in connection with a variance analysis (body and organ weight data)

Analysis of variance followed by Dunnett test (Erythrocytes, Hemoglobin, Hematocrit, Mean Corpuscular Hemoglobin, Mean Corpuscular Hemoglobin Concentration, Mean Corpuscular Volume Erythrocytes, Thrombocytes, Albumin, Creatinine, Chloride, Glucose, Protein, Triglycerides, Urea)

Kruskal-Wallis-Test with a Steel-Test (food and water intake data)

Kruskal-Wallis test followed by adjusted U test (Heinz Bodies, Hepato Quick, Leucocytes, Reticulocytes, Band NeutrophiIs, EosinophiIs, Lymphocytes, Monocytes, Segmented Neutrophils, Bilirubin total, Gamma-Gtutamyltransferase, Specific Gravity, Glucose, dipstick, pH, dipstick, Protein Quantitative, Volume, pH)

Adjusted Welsh test (Atanine aminotransferase, Alkaline phosphatase, Aspartate aminotransferase, Choiinesterase, Choiinesterase in Erythrocytes, Choiinesterase in Brain, Cholesterol, Calcium, Glutamate dehydrogenase, Potassium, Sodium, Inorganic Phosphate)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
All dose groups: a higher mortality in females was evident from Week 52 onwords; dose correlation is lacking

For details please refer to Table 1 in "any other information on results incl. tables".
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
3000/4000 ppm: body weight depression in males from Week 7 / in females from Week 13 onwords

For details please refer to Table 2 in "any other information on results incl. tables".
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
3000/4000 ppm: slightly elevated food intake per kg body weight in both sexes (12.3%/19.2%)
The test substance intake in the treatment groups roughly corresponds to the theoretical dose intervals.

For details please refer to Table 3 in "any other information on results incl. tables".
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
3000/4000 ppm: indication for slightly reduced food efficiency in both sexes

For details please refer to Table 3 in "any other information on results incl. tables".
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
no effects observed

For details please refer to Table 4 in "any other information on results incl. tables".
Ophthalmological findings:
effects observed, treatment-related
Description (incidence and severity):
1000 and 3000 ppm: higher incidence of moderate or severe opacities in the (whole) lens cortex of males at termination

For details please refer to Table 5 in "any other information on results incl. tables".
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
3000/4000 ppm: decreased Hb and Ht in males in Week 26/27 only, decreased MCH and MCHC in females

For details please refer to Tables 6 and 7 in "any other information on results incl. tables".
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
3000/4000 ppm: slightly increased gamma glutamyltransferase (GOT) activity in both sexes), but not at all dates and without relevant time-dependent increase
Slightly decreased ASAT and APh activities in animals dosed at 1000 ppm and higher are not considered to indicate an adverse effect, since the values were within the physiological range.

For details please refer to Table 8 in "any other information on results incl. tables".
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Quantitative urinalyses did not reveal a treatment-related effect on pH, sediment, proteinurea, urine volume or urine density. Significant deviations from control values in these parameters were not considered relevant, since dose correlations were lacking.

For details please refer to Tables 10 and 11 in "any other information on results incl. tables".
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):
Interim necropsy:
3000/4000 ppm: significantly increased liver weights (absolute and relative ) in both sexes
Terminal necropsy:
3000/4000 ppm: partly significantly increased liver weights (absolute and relative) in both sexes, slightly decreased spleen weights (absolute and relative) in both sexes, increased relative testis weights in males

For details please refer to Tables 12 and 13 in "any other information on results incl. tables".
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Interim necropsy:
no effects observed
Terminal necropsy:
3000/4000 ppm: 6 males and 5 females appeared skinny
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Interim examinations:
200 ppm: diffuse hyperplasia in the transitional epithelium of the urinary bladder in males
1000 ppm: diffuse hyperplasia in the transitional epithelium of the urinary bladder in males, hepatocellular hypertrophy (centrilobular) in both sexes
3000/4000 ppm: diffuse hyperplasia in the transitional epithelium of the urinary bladder, hepatocellular hypertrophy (centrilobular) and slight increase in the incidence and severity of vacuolation of zona fasciculata cells in the adrenal cortex in both sexes

For details please refer to Table 14 in "any other information on results incl. tables".

Terminal examinations:
1000 ppm: centrilobular hepatocytes with cytoplasmic changes and/or hypertrophy more frequently and decreasing tendency for pituitary hyperplasia as well as for degenerative alterations in the adrenals, tongue, heart and kidney in females
3000/4000 ppm: eosinophilic foci, focal degenerative changes (periportal) and cytoplasmic changes of hepatocytes (periportal and centrilobular) in the liver of males, peliotic foci, single cell necrosis and cytoplasmic changes of hepatocytes (centrilobular) and/or hypertrophy in the liver of females;
increased number of animals with a focal or diffuse simple transitional hyperplasia in the urinary bladder, increases of Proliferating Cell Nuclear Antigen (PCNA) labelling index of the urinary bladder in the hyperplastic segment of the transitional epithelium of females;
increased incidence of follicular hyperplasia (males) and mineralization of the follicular colloid (both sexes) in thyroid glands;
increased number of males with increase in vacuolation of cells in the zona fasciculata of the adrenal glands;
higher frequency of atrophy and/or fibre degenerations of the skeletal muscle in females;
markedly higher incidence and degree of degenerative myelinopathy in the sciatic nerve in both sexes;
decreasing tendency for pituitary hyperplasias as well as for degenerative alterations in the adrenals, tongue, heart and kidney in females;
dilated glands in the stomach mucosa and increase in pigment storage in the spleen of females

For details please refer to Table 15 in "any other information on results incl. tables".
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Interim examinations:
In animals scheduled for interim kill, a no dose-related occurrence of a tumour type was evident.
Terminal examinations:
1000 ppm: adenomas in the thyroid follicles of one male
3000/4000 ppm: one transitional cell carcinoma and two transitional cell papillomas in the urinary bladder of females and one transitional carcinoma in the urethra of a male;
adenomas/carcinoma in the thyroid follicles of males (incidence per increasing dose group: 0-0-0-1/0-2/1)
For details please refer to Tables 16 to 19 in "any other information on results incl. tables".
Other effects:
effects observed, treatment-related
Description (incidence and severity):
ChE activity plasma
No relevant treatment-related effects observed.

ChE activity erythrocytes
1000 ppm: significantly reduced ChE activity in both sexes
3000/4000 ppm: significantly reduced activity in both sexes

ChE activity brain
1000 ppm: reduced ChE activity in females
3000/4000 ppm: reduced ChE activity in both sexes

For details please refer to Table 9 in "any other information on results incl. tables".
Details on results:
Mortality:
The number of males which had died unscheduled was equally distributed among the groups up to 3000 ppm. In all female treatment groups a higher mortality is evident from Week 52 onwards. Since a dose correlation is lacking in the wide dose range (50 and 4000 ppm) a treatment effect is not assumed. Neither for males nor for females did the global comparison of the survival curves (Wilcoxon Test) gave any indication of statistically relevant differences between the groups (p = 0.5782).

Body weight and weight changes:
There were no significant and dose-dependent effects on body weights up to 1000 ppm in males. Males ingesting 3000 ppm gained less body weight from week 7 onwards with a maximum difference of 11.0% in Week 105. The differences were significant in most cases. In female rats there is also no toxicologically relevant effect on body weights up to the dose of 1000 ppm. The few significantly lower means at 1000 ppm (between Weeks 43 to 61) are not interpreted as an adverse effect, since deviations to control data are very small (maximal 6.3% in Week 51). At 4000 ppm a significant (from Week 13 onwards) body weight depression was observed with a maximum of 14.3% in Week 85.

Food consumption and compound intake:
The food intake per animal at doses of up to 3000 ppm (males) or 4000 ppm (females) was comparable to that of untreated rats. The slightly elevated intake data per kg body weight in rats receiving 3000 (12.3%) or 4000 ppm (19.2%) could be interpreted as an indication of a slightly reduced feed efficiency.

Opthalmological findings:
Males of the 1000 and 3000 ppm groups showed a higher incidence of moderate or severe opacities in the (whole) lens cortex. The highest frequency of lenses showing slight (wedge shaped) waterclefts, being a pre-stage of cortical opacities, was noted in control males. This incidence distribution indicates a shift of lens waterclefts towards cortical opacities in 1000 and 3000 ppm males. In 4000 ppm females no remarkable lens findings could be detected compared to controls. Concerning the remaining eye compartments no treatment effect is visible.

Haematological findings:
There were no toxicologically relevant changes in the erythrocyte parameters (count, MVC, MCHC and MCH), in the haemoglobin concentration or in the haematocrit in males or females up to 1000 ppm. Some significant differences in these groups and parameters are not considered as a sign of toxicity, since deviations to control values were very small (MCV, MCH and elevated MCHC means in treated males in Week 26/27) and/or a dose correlation was lacking (erythrocyte counts). The haemoglobin concentration and haematocrit values were significantly reduced in 3000 males at Week 26/27, but not thereafter. Other significant changes in this group were slight and without corroborating findings. In 4000 ppm females no significant changes were seen with regard to haemoglobin concentration, haematocrit and erythrocyte counts but the calculation of MCH and MCHC revealed consistently lower (partly p<0.01) values than in controls and remaining groups. Analysis of erythrocyte morphology did not reveal abnormalities that could be regarded as a toxic effect. No relevant changes in platelet counts were found in treated males and females. The very slight increase in the means of platelet count of 4000 ppm females in Weeks 26/27 and 52 do not reflect an adverse effect, since individual values were within the reference range. The means of the thromboplastin time in the treatment groups were not remarkably different from those at 0 ppm. No changes of reticulocytes were observed up to the highest dose level in both sexes. No toxicologically relevant changes in leukocyte counts and differential blood counts were visible in the dose range investigated.

Clinical biochemistry findings:
Neither male nor female rats showed any remarkable changes in the plasma activity of aspartate aminotransferase (ASAT), alkaline phosphatase (APh) and glutamate dehydrogenase (GLDH) up to 200 ppm. At higher doses there was a tendency towards slightly (sometimes significantly) lower aspartate aminotransferase (ASAT) and alkaline phosphatase (APh) activities. These changes were not considered to indicate an adverse effect, since the values were within the physiological range. The activity of the alanine aminotransferase (ALAT) was unchanged in all treatment groups. A trend towards slightly elevated gamma glutamyltransferase (GOT) activity was at 3000 or 4000 ppm (p<0.01), but not at all dates and without relevant time dependent increase.

Urinalysis findings:
In Week 98/99 quantitative pH measurements were performed by a microelectrode. Results of these determinations do not indicate any influence of the test substance on the urine pH in the dose range investigated. Scanning-electron-microscopy of urine sediment did not reveal any indication of a change in amount or morphology of urine crystals due to the treatment. Quantitative urinalyses did not reveal a treatment-related effect on proteinurea, urine volume or urine density. Significant deviations from control values in these parameters were not considered relevant, since dose correlations were lacking.

Organ weight findings including organ body weight ratios:
At interim necropsy all absolute and relative organ weights except the liver weights were comparable with those of controls. A few significant changes can be attributed to differences in body weight. The liver weights were not remarkably changed up to 1000 ppm. In 3000 ppm males they were significantly increased by 18.5% (absolute) and 17.6% (relative), whereas in 4000 ppm females the increase was 18.8% (absolute) and 41.7% (relative). At terminal necropsy there were no toxicologically relevant differences between the dose groups and the controls with respect to the weights of the brain, adrenals, heart, kidneys or ovaries. The few differences marked as significant in these organs were very small and attributable to varying body weights. The liver weights were inconspicuous up to 1000 ppm. At higher concentrations absolute and relative liver weights were increased (partly p<0.01). The relative means were 20.2% ( 3000 ppm males) or 32.9% (4000 ppm females) above the corresponding control value. Up to 1000 ppm spleen and testis weights were unremarkable. At the top concentration levels slightly lower (up to -23%) spleen weights (absolute and relative) were seen in both sexes and the relative testes weights were elevated (p<0.01) by about 17%. These deviations were partly significant.

Gross pathological findings:
No treatment-related macroscopical findings were observed in rats scheduled for a treatment period of 12 months. Gross-pathological findings recorded at the necropsies performed during the in life phase in case of unscheduled deaths or at the end of the study revealed no evidence of dose-related organ lesions in any of the groups up to and including doses of 3000 or 4000 ppm. There were six 3000 ppm males and five 4000 ppm females that appeared skinny at necropsy.

Histopathological findings (non-neoplastic):
In animals scheduled for a 12-months treatment revealed test substance-related lesions in the adrenals, liver and urinary bladder. In the adrenal cortex a slight increase in the incidence and severity of vacuolation of the zona fasciculata cells was recorded at 3000 or 4000 ppm. A minimal to moderate hepatocellular hypertrophy, predominantly centrolobular, was noted in rats at 1000 ppm and above.
In the urinary bladder a diffuse hyperplasia (minimal to slight) was observed in the transitional epithelium at 200 ppm and above (males) or in the group 4000 ppm (females). For the lesions in the urinary bladder, liver and adrenals a statistically significant trend was established. All other microscopical findings recorded were considered to be incidental. In animals scheduled for terminal kill, there were no treatment-related non-neoplastic lesions at concentrations of up to 1000 ppm in males and 200 ppm in females. In the liver of 3000 ppm males eosinophilic foci, focal degenerative changes (periportal) and cytoplasmic changes of hepatocytes (periportal and centrilobular) were noted with a markedly higher incidence than in the other groups. These findings were associated with a reduced number of clear cell foci at 3000 ppm. In males of the groups 0, 50, 200 and 1000 ppm and in all treated females the frequency of all these findings did not exceed the reference range of rats of this age. In females centrilobular hepatocytes with cytoplasmic changes and/or hypertrophy were detected more frequently from 1000 ppm onwards. Additionally, in 4000 ppm females peliotic foci and single cell necrosis were noted with increased incidences. Most of the liver lesions mentioned were found to show a statistically significant positive trend. The number of animals with a focal or diffuse simple transitional hyperplasia in the urinary bladder was increased (trend mostly p<0.01) in high dose males and females. In 4000 ppm females atrophy (p<0.01) and/or fibre degenerations (p<0.05) of the skeletal muscle were found with a higher frequency than in the other groups. In 3000 ppm males and 4000 ppm females a degenerative myelinopathy was detected in the sciatic nerve with a markedly higher incidence (trend p<0.01) and a higher degree than in the other groups, where the frequency of this lesion did not exceed the range normally found in old rats. In the thyroid glands the incidence of follicular hyperplasia (males) and mineralization of the follicular colloid (males and females) was significantly increased in 3000/4000 ppm rats. At 3000 ppm there were more (trend p<0.01) males exhibiting an increase in vacuolation of cells in the zona fasciculata of the adrenal glands. Statistically significant increases of proliferating cell nuclear antigen (PCNA) labeling index of the urinary bladder were only achieved in the hyperplastic segment of the transitional epithelium of 4000 ppm females. In the bladder of 1000 ppm females and in the non-hyperplastic bladder segment of the 4000 ppm rats the calculated values were similar to those of control females.The remaining observed findings were considered to be secondary possibly due to retarded aging, which is often seen in toxicological studies, if body weight is reduced as in the present study. These findings included a decreasing tendency for pituitary hyperplasia as well as for degenerative alterations in the adrenals, tongue, heart and kidney essentially in 1000 and 4000 ppm females. The elevated (p<0.05) number of 4000 ppm females showing dilated glands in the stomach mucosa was considered unspecific and not treatment-related. Also the increase in pigment storage in the spleen (p<0.01) was not considered treatment-related.

Histpathological findings (neoplastic):
In animals scheduled for interim kill, a no dose-related occurrence of a tumour type was evident. At the terminal kill, one transitional cell carcinoma and two transitional cell papillomas (trend p<0.01) were detected in the urinary bladder of 4000 ppm females, whereas no bladder tumour occurred in the remaining female groups or in males. Additionally, one transitional carcinoma was seen in the urethra of a 3000 ppm male. In thyroid follicles of males adenomas were observed with a frequency of 0-0-0-1-2 (trend p<0.05) and carcinomas with an incidence of 0-0-0-0-1. Uterine adenocarcinomas were noted in 4000 ppm females more often (trend p<0.01) than in the other groups. Additionally, there was one squamous cell carcinoma (p<0.05) in this group. However, the incidences of both tumour types do not exceed the historical control range and are therefore, considered to be not compound-induced. Decreased tumour incidences were noted for the mammary fibroadenomas(15-10-7-6-5) and pituitary adenomas (24-18-24-14-11) in the female treatment groups. The incidences of the remaining tumours were not distributed in a treatment-related manner and comparable with those known from own historical control collectives. The number of cases, in which a tumour type occurred in one group only was comparable in the 3000/4000 ppm and 0 ppm group (for details please refer to Tables 16 in "any other information on results incl. tables".). The overall tumour incidence and the total number of benign and malignant neoplasms per group were not increased in treated males and females at all concentrations (for details please refer to Table 17 in "any other information on results incl. tables"). The number of malignant tumours was even reduced in males receiving 200 ppm and above. No remarkable differences were observed deaths in the number of tumour bearing rats or animals with a benign, malignant or benign and malignant tumour between the control and treatment groups for both, intercurrent or scheduled deaths (for details please refer to Table 18 in "any other information on results incl. tables"). As regards the time of occurrence of the tumours, there were no significant differences between treated rats and controls (for details please refer to Table 19 in "any other information on results incl. tables").

Other findings: Cholinesterase activities (ChE)
No significant ChE inhibition occurred in the plasma. The 24% lower plasma ChE activity of 3000 ppm males (Week 78) is not regarded as toxicologically relevant, because this was an isolated event, where in the control group relatively high activities were measured compared to those of the other time points. In addition, plasma ChE activity was even not affected when e.g. ChE activity in erythrocytes was inhibited by 90%. No remarkable ChE inhibition in erythrocytes were detected in males and females up to 200 ppm. At higher concentrations a dose-dependently and significantly lower ChE activity (inhibited up to 82% in males and 90% in females) was measured compared to control means. The significantly lower activity in 200 ppm females in Week 52/53 is considered to be incidental, because activity at this dose level was not remarkably inhibited at the other three time points, where stronger effects were noted in the two highest dose groups. Also in Week 52/53, a relatively high activity in controls may have contributed. Up to 3000 ppm (males) and 200 ppm (females) no significant or biologically relevant effect was noted on the brain ChE activity. In females there was a significantly reduced activity at 1000 and 4000 ppm (up to 41%). The inhibition values of 13 and 17% in 1000 ppm females and those of 14% calculated for 3000 ppm males at termination might be of questionable toxicological relevance.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
200 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to this dose level
Remarks on result:
other: corresponding to: 10.3 mg/kg bw/day (males) / 14.6 mg/kg bw/day (females)
Key result
Dose descriptor:
LOAEL
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
other: Cholinesterase activity in erythrocytes and brain
Remarks on result:
other: corresponding to: 52.7 mg/kg bw/day (males) / 75.4 mg/kg bw/day (females)

Target system / organ toxicity

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 ppm
System:
nervous system
Organ:
other: cholinesterase activity inhibited in erythrocytes (1000 ppm and above) and brain (4000 ppm) (statistically significant inhibition by 20% or more in erythrocytes or brain is considered a clear toxicological effect)
Treatment related:
yes
Dose response relationship:
yes

Any other information on results incl. tables

Table 1: Cumulative Mortality data (number of dead animals up to week indicated)

Dose (ppm)

0

50

200

1000

3000

sex (male)

Weeks 0-52 (n = 60)

Week 26

0

1

0

0

0

Week 53

0

1

0

2

1

Weeks 53-104 (n = 50)

Week 79

5

4

1

5

1

Week 104

19

17

21

17

13

Dose (ppm)

0

50

200

1000

4000

sex (female)

Weeks 0-52 (n = 60)

Week 26

0

0

0

0

0

Week 53

0

2

1

0

2

Weeks 53-104 (n = 50)

Week 79

3

7

8

8

9

Week 104

13

19

22

20

21

 

Table 2: Body weights [g]

sex

m

m

m

m

m

f

f

f

f

f

Dose (ppm)

0

50

200

1000

3000

0

50

200

1000

4000

0

1

2

3

4

5

6

7

8

9

10

11

12

13

15

17

19

21

23

25

27

29

31

33

35

37

39

41

43

45

47

49

51

53

55

57

59

61

63

65

67

69

71

73

75

77

79

81

83

85

87

89

91

93

95

97

99

101

103

105

155

203

239

272

299

322

336

362

378

382

393

403

412

427

435

456

463

471

472

477

482

492

492

502

500

510

515

519

509

517

519

523

526

531

532

535

535

542

542

542

543

553

552

553

558

550

551

558

555

559

554

553

550

549

550

553

551

544

542

543

156

203

243

276

304

325

344

362

378

394

406

417

428

436

448

465

468

479

480

486

494

498

503

509

507

515

518

521

519

523

521

528

532

530

531

534

534

536

540

539

541

548

549

548

550

552

543

551

549

548

542

537

529

530

529

534

530

520

508 +

511

155

203

245

278

306

329

348

364

378

395

406

417

423

430

444

459

463

470

480

487

493

495

503

507

509

508

514

516

515

518

520

524

529

533

534

538

539

539

541

541

542

547

545

541

544

539

536

536

538

532

520 +

522

513 +

527

528

522

519

510 +

514

529

154

200

241

275

301

326

341

356

368

385

401

400

417

420

438

448

455

464

472

480

483

488

496

495

502

509

507

510

512

512

513

519

522

525

527

530

534

533

532

538

542

549

547

547

550

550

552

556

555

553

549

547

533

542

535

531

528

523

518

515

152

189++

238

264

289

312

330

344++

360++

372

382

391

401

401++

421

427++

441+

449+

454

463

466

468+

480

479+

485

483++

486++

489++

492

490++

490++

496++

500++

502++

505+

506++

513

511++

512++

515+

516+

521++

519++

516++

518++

512 ++

523 +

524 ++

527 +

526 +

518 +

513 ++

493 ++

504 ++

498 ++

497 ++

492 ++

495 ++

491 ++

483 ++

130

149

163

176

186

196

205

212

217

222

225

229

234

242

242

247

249

255

258

260

261

267

271

277

276

280

284

286

289

290

295

296

301

302

304

309

311

312

314

315

319

323

322

325

329

333

328

333

333

336

327

327

329

327

326

328

323

323

318

313

127

145

161

173

183

191

200

208

212

216

222

226

230

234

240

243

247

251

254

257

257

261

266

271

273

275

279

281

284

285

289

288

291

291

294

298

300

303

307

307

309

316

314

318

321

323

321

329

330

331

326

325

324

324

325

327

325

324

315

325

130

150

166

179

190

199

207

211

217

223

228

232

238

239

244

249

252

258

260

263

264

266

272

276

280

281

285

288

289

289

293

293

297

302

304

307

309

308

312

315

316

322

322

325

327

328

327

332

333

337

332

330

331

327

329

326

325

320

312

341

128

147

164

178

186

196

203

207

213

219

221

228

231

234

237

242

246

249

252

254

257

257

261

266

270

270

274

276

276+

275+

280+

279++

282++

284+

287+

291+

292+

294+

300

301

303

308

307

311

313

314

310

320

320

320

316

314

317

317

313

311

311

312

307

320

130

147

167

179

189

196

202

205

209

215

217

222

225

226++

230+

234++

234++

238++

242++

245++

246++

248++

252++

253++

257++

258++

262++

264++

264++

263++

266++

266++

268++

270++

271++

274++

275++

279++

278++

280++

281++

285++

283++

283++

286++

284 ++

284 ++

289 ++

291 ++

288 ++

286 ++

288 ++

287 ++

289 ++

289 ++

286 ++

283 ++

284 ++

281 ++

295

+ difference against control for p<0.05

++ difference against control for p<0.01

 

Table 3: Mean daily and cumulative intake of food and test compound

Intake food

 

 

 

g/animal

g/kg bw

Dose (ppm)

Sex

Days

Total

per Day

Total

per Day

0

m

708

14715

20.8

35813

50.6

50

m

708

14923

21.1

35653

50.4

200

m

708

15169

21.4

36331

51.3

1000

m

708

15391

21.7

37307

52.7

3000

m

708

15807

22.3

40218

56.8

0

f

708

12072

17.1

48515

68.5

50

f

708

12522

17.7

51527

72.8

200

f

708

12729

18.0

51524

72.8

1000

f

708

12712

18.0

53372

75.4

4000

f

708

13177

18.6

57821

81.7

Intake of test compound

 

 

 

mg/animal

mg/kg bw

Dose (ppm)

Sex

Days

Total

per Day

Total

per Day

50

m

708

746

1.1

1783

2.5

200

m

708

3034

4.3

7266

10.3

1000

m

708

15391

21.7

37307

52.7

3000

m

708

47421

67.0

120654

170.4

50

f

708

626

0.9

2576

3.6

200

f

708

2546

3.6

10305

14.6

1000

f

708

12712

18.0

53372

75.4

4000

f

708

52707

74.4

231285

326.7

 

Table 4: Mean daily cumulative water intake

Intake of water

 

 

 

g/animal

g/kg bw

Dose (ppm)

Sex

Days

Total

per Day

Total

per Day

0

m

708

18267

25.9

40827

57.8

50

m

708

18971

26.9

41396

58.6

200

m

708

19254

27.3

42187

59.8

1000

m

708

19034

27.0

42158

59.7

3000

m

708

19391

27.5

44915

63.6

0

f

708

18478

26.2

67633

96.8

50

f

708

18745

26.6

70078

99.3

200

f

708

18848

26.7

68707

97.3

1000

f

708

18539

26.3

70478

99.8

4000

f

708

17344

24.6

69761

98.8

 

Table 5: Ophthalmological findings at termination (Incidences in %)

sex

m

m

m

m

f

f

Dose (ppm)

0

200

1000

3000

0

4000

No. of eyes examined

61

71

61

73

76

58

Turbid cornea, including vascularisation of cornea

1.6

7.0

1.6

8.2

0

3.4

Turbid cornea, including vascularisation of cornea

Snow ball like turbidities between lens and vitreous

body (= posterior lenticular rupture)

11.4

4.2

8.2

13.7

7.9

0

Diffuse retrolenticular opacity (all degrees)

24.6

9.8

18.0

17.8

10.5

8.6

Wedge shaped waterclefts in the cortex, slight

18.0

7.0

8.2

8.2

3.9

5.1

Wedge shaped waterclefts in the cortex, moderate

0

0

3.3

1.4

0

1.7

Lens opacity in the whole cortex, slight

1.6

1.4

0

2.7

2.6

1.7

Lens opacity in the whole cortex, moderate

1.6

1.4

1.6

5.5

3.9

0

Lens opacity in the whole cortex, severe

1.6

0

6.5

5.5

6.6

3.4

 

Table 6: Hematology

Dose

 

 

LEU

ERY

HB

HCT

MCV

MCH

MCHC

RETI

HEINZ

THRO

HQUICK

ppm

Sex

Week

109/L

109/L

g/L

L/L

fl

pg

g/LERY

0/00

0/00

109/L

sec

0

m

26/27

10.3

9.04

147

0.459

50.8

16.3

320

18

0

1040

25.4

50

m

26/27

8.7

8.91

147

0.453

50.9

16.5

324

19

0

998

25.2

200

m

26/27

8.3

8.51

141

0.439

51.6

16.5

320

18

0

1109

24.7

1000

m

26/27

8.3

9.06

141

0.434

48.0++

15.6

325

17

0

1053

26.2

3000

m

26/27

8.0

8.58

138+

0.425++

49.6

16.1

325

17

1

982

25.4

0

m

52

9.5

9.42

153

0.487

51.8

16.2

313

16

0

1082

26.3

50

m

52

8.5

9.17

149

0.479

52.3

16.3

312

17

0

1059

26.0

200

m

52

8.4

8.87+

148

0.471

53.1

16.7

314

18

0

1172

26.1

1000

m

52

7.4

9.37

146

0.462

49.4

15.6

315

20

0

1082

25.4

3000

m

52

8.2

9.29

146

0.472

50.8

15.7

309

20

0

1026

26.3

0

m

78

8.0

9.04

151

0.476

52.7

16.8

318

17

0

1091

24.7

50

m

78

7.3

8.99

149

0.472

52.5

16.6

316

14

0

1073

24.6

200

m

78

6.7

8.20+

141

0.441

53.7

17.2

319

16

0

1277

24.0

1000

m

78

6.4

8.93

141

0.441

49.5+

15.9+

321

17

0

1041

24.1

3000

m

78

7.5

8.77

139

0.440

50.2

15.9+

316

15

0

998

24.1

0

m

103/104

9.2

9.26

160

0.512

55.4

17.3

312

20

0

1220

25.9

50

m

103/104

7.9

8.63

148

0.457+

52.9

17.1

322++

27

0

1158

25.0

200

m

103/104

7.9

8.85

154

0.473

53.5

17.5

326++

16

0

1156

24.2

1000

m

103/104

7.3

8.73

144

0.443++

50.9++

16.5

324++

23

0

1186

24.8

3000

m

103/104

6.9

9.07

150

0.466

51.5++

16.5

321+

29

0

1081

25.8

0

f

26/27

6.0

8.21

143

0.439

53.5

17.4

325

15

0

915

24.5

50

f

26/27

6.2

7.94

136

0.416

52.4

17.1

328

15

0

978

23.7

200

f

26/27

5.9

8.10

139

0.422

52.2

17.2

330

14

0

979

23.8

1000

f

26/27

6.2

8.01

137

0.424

52.8

17.1

325

17

0

955

23.8

4000

f

26/27

5.8

8.57

139

0.434

50.6++

16.2++

320

17

0

1046++

22.4

 

0

f

52

4.6

8.03

140

0.440

54.8

17.4

318

20

0

809

24.7

50

f

52

5.8

8.15

138

0.437

53.6

17.0

317

18

0

921

25.4

200

f

52

5.7

8.26

142

0.440

53.4

17.2

322

17

0

956

25.2

1000

f

52

5.7

8.25

140

0.447

54.2

17.0

314

19

0

897

24.1

4000

f

52

5.8

8.36

136

0.440

53.1

16.4+

309++

20

0

1017+

23.8

0

f

78

4.1

8.33

146

0.454

54.6

17.5

321

25

0

858

23.2

50

f

78

4.6

7.79

136

0.429

56.6

17.8

316

44

0

795

23.5

200

f

78

5.6

8.24

142

0.443

53.8

17.3

321

31

0

840

23.4

1000

f

78

4.3

8.16

143

0.451

55.4

17.6

317

31

0

848

26.2++

4000

f

78

4.7

8.06

133

0.431

53.6

16.6

309++

36

0

930

24.8+

0

f

103/104

5.6

8.29

151

0.453

54.9

18.3

333

31

0

1003

24.0

50

f

103/104

4.7

8.34

151

0.454

54.5

18.1

333

28

0

984

23.7

200

f

103/104

6.8

8.22

149

0.450

54.7

18.1

330

26

0

994

24.6

1000

f

103/104

4.3

8.33

151

0.463

55.7

18.1

325

29

0

948

24.8

4000

f

103/104

5.1

8.22

143  

0.443

 54.2 

17.4 

322+ 

 31 

0 

1066 

23.9 

+ difference against control for p<0.05

++ difference against control for p<0.01

 

Table 7: Differential blood count

Dose

Week

Sex

LYM

SEGM

EOS

MONO

BAND

ppm

 

 

%

%

%

%

%

0

26/27

m

87.5

8.0

0.2

4.3

0.0

50

26/27

m

89.8

6.5

0.3

3.4

0.0

200

26/27

m

86.0

10.1

0.6

3.3

0.0

1000

26/27

m

87.9

8.1

0.7

3.3

0.0

3000

26/27

m

89.4

7.6

0.6

2.5

0.0

0

52

m

79.7

12.7

1.0

6.6

0.0

50

52

m

78.8

11.7

1.8

7.7

0.0

200

52

m

79.1

12.7

0.9

7.3

0.0

1000

52

m

77.5

13.6

1.4

7.6

0.0

3000

52

m

78.0

13.7

0.6

7.8

0.0

0

78

m

78.4

13.9

1.5

6.2

0.0

50

78

m

75.9

16.2

2.2

5.6

0.0

200

78

m

74.9

17.4

1.7

6.2

0.0

1000

78

m

74.7

18.9

1.1

5.3

0.0

3000

78

m

77.6

17.0

0.8

4.6

0.0

0

103/104

m

67.8

23.5

1.2

7.4

0.0

50

103/104

m

62.7

29.7

1.7

6.0

0.0

200

103/104

m

70.0

23.6

1.5

4.8

0.1

1000

103/104

m

66.6

25.4

2.6

5.5

0.0

3000

103/104

m

71.8

23.0

1.2

4.2

0.0

0

26/27

f

88.4

8.4

0.8

2.5

0.0

50

26/27

f

86.0

11.3

0.8

1.9

0.0

200

26/27

f

89.5

7.8

0.8

1.9

0.0

1000

26/27

f

90.7

8.2

0.4

0.7+

0.0

4000

26/27

f

88.7

8.5

0.8

2.0

0.0

0

52

f

79.9

14.0

1.4

4.7

0.0

50

52

f

76.5

16.5

1.0

6.0

0.0

200

52

f

80.7

13.3

1.2

4.8

0.0

1000

52

f

80.5

15.0

0.6

3.9

0.0

4000

52

f

86.4

9.7

0.5

3.4

0.0

0

78

f

71.8

23.7

1.2

3.3

0.0

50

78

f

75.7

16.2

1.7

6.4

0.1

200

78

f

65.9

26.9

1.0

6.3

0.0

1000

78

f

73.5

19.8

1.2

5.6

0.0

4000

78

f

78.4

16.0

1.1

4.5

0.0

0

103/104

f

70.4

27.4

1.2

1.1

0.0

50

103/104

f

69.3

28.8

0.8

1.1

0.0

200

103/104

f

60.4

37.3

1.3

1.0

0.0

1000

103/104

f

75.8

22.0

1.0

1.2

0.0

4000

 103/104

f

73.1

 23.8

 1.6

 1.6

0.0

+ difference against control for p<0.05

++ difference against control for p<0.01

 

Table 8: Clinical Chemistry

Dose

Week

Sex

ASAT (GOT)

ALAT (GPT)

APh

GGT

GLDH

ppm

 

 

U/L

U/L

U/L

U/L

U/L

0

26/27

m

41.3

38.9

190

1

5.7

50

26/27

m

38.8

40.1

186

1

5.4

200

26/27

m

44.4

40.3

171

2

7.9

1000

26/27

m

38.5

40.4

157+

2

5.0

3000

26/27

m

35.1

37.7

150+

3++

3.8

0

52

m

39.9

42.2

184

1

16.2

50

52

m

40.5

44.l

193

1

22.8

200

52

m

36.0

37.9

162

2

12.0

1000

52

m

31.2

37.4

154+

1

12.6

3000

52

m

34.6

41.0

156+

2

12.8

0

78

m

38.8

36.5

157

1

16.0

50

78

m

42.5

43.8

178

2

22.0

200

78

m

35.1

32.1

142

1

14.0

1000

78

m

33.2

36.0

141

1

17.9

3000

78

m

32.7

35.7

144

2

20.0

0

103/104

m

40.2

37.5

164

3

21.4

50

103/104

m

42.2

33.1

175

2

21.6

200

103/104

m

37.4

32.3

134

2

10.8

1000

103/104

m

37.4

29.8

137

3

14.8

3000

103/104

m

36.9

35.3

140

3

19.0

0

26/27

f

40.7

43.4

121

0

11.5

50

26/27

f

48.0

43.6

133

0

18.7

200

26/27

f

41.0

44.9

122

0

11.3

1000

26/27

f

32.3+

41.9

103

0

6.8

4000

26/27

f

30.9++

42.6

105

1

1.0

0

52

f

44.7

42.6

120

0

17.2

50

52

f

46.8

44.1

134

0

22.9

200

52

f

42.5

43.8

111

0

20.4

1000

52

f

55.4

55.2

99

0

63.3

4000

52

f

33.3

45.0

118

0

9.2

0

78

f

59.4

45.7

100

1

59.2

50

78

f

89.4

62.2

113

0

104.8

200

78

f

73.9

54.4

91

0

87.7

1000

78

f

40.3

36.3

81+

1

29.1

4000

78

f

41.2

37.5

90

2+

18.4

0

103/104

f

61.3

45.9

114

0

35.5

50

103/104

f

61.3

49.0

114

1

53.7

200

103/104

f

76.6

53.8

102

0

70.9

1000

103/104

f

45.7

41.2

92

0

26.4

4000

103/104

f

 43.4

 43.6 

102 

2++ 

41.5 

+ difference against control for p<0.05

++ difference against control for p<0.01

 

Table 9: Activities and inhibition of cholinesterases (CHE)

Dose

Week

Sex

CHE/Brain

CHE/Plasma

CHE/Erythrocytes

ppm

 

 

U/g

% Inhibition

kU/L

% Inhibition

kU/L

% Inhibition

0

27

m

-

 

0.50

 

1.06

 

50

27

m

-

 

0.52

 

1.06

 

200

27

m

-

 

0.52

 

0.88

17

1000

27

m

-

 

0.50

 

0.65+

39

3000

27

m

-

 

0.45

10

0.32++

70

0

52/53

m

10.64

 

0.62

 

1.14

 

50

52/53

m

10.46

2

0.68

 

1.15

 

200

52/53

m

10.56

 

0.63

 

1.01

11

1000

52/53

m

10.26

4

0.61

2

1.00

12

3000

52/53

m

9.54

10

0.56

10

0.57++

50

0

78

m

-

 

0.72

 

1.24

 

50

78

m

-

 

0.71

 

1.35

 

200

78

m

-

 

0.71

1

1.16

6

1000

78

m

-

 

0.63

13

0.84+

32

3000

78

m

 

 

0.55

24

0.55++

55

0

103/104/105

m

9.88

 

0.66

 

0.83

 

50

103/104/105

m

9.87

 

0.95

 

0.76

8

200

103/104/105

m

9.86

 

0.86

 

0.70

16

1000

103/104/105

m

9.47

4

0.65

2

0.50++

40

3000

103/104/105

m

8.50

14

0.55

17

0.15++

82

0

26/27

f

-

 

2.47

 

0.77

 

50

26/27

f

-

 

2.24

9

0.83

 

200

26/27

f

-

 

2.15

13

0.70

9

1000

26/27

f

-

 

2.57

 

0.38++

51

4000

26/27

f

-

 

2.30

7

0.08++

90

0

52/53

f

11.28

 

2.39

 

1.24

 

50

52/53

f

10.82+

4

2.21

8

1.17

6

200

52/53

f

10.80

4

2.39

 

0.96+

23

1000

52/53

f

9.41++

17

2.73

 

0.58++

53

4000

52/53

f

7.07++

37

2.49

 

0.34++

73

0

78

f

-

 

2.18

 

1.19

 

50

78

f

-

 

2.03

7

1.18

1

200

78

f

-

 

2.12

3

1.01

15

1000

78

f

-

 

2.45

 

0.45++

62

4000

78

f

-

 

2.22

 

0.13++

89

0

103/104/105

f

10.22

 

2.25

 

0.74

 

50

103/104/105

f

10.02

2

2.31

 

0.66

11

200

103/104/105

f

9.86

4

1.95

13

0.73

1

1000

103/104/105

f

8.91++

13

2.11

6

0.28++

62

 4000

103/104/105

f

6.02++

41

 2.08

 8

0.08++

89

+ difference against control for p<0.05

++ difference against control for p<0.01

 

Table 10: Determination of urine pH week 98/99

 

pH mean value

Sex

male

female

Dose (ppm)

0

7.55

7.20

50

7.57

7.47

200

7.32

7.12

1000

7.45

7.64

3000

7.34

-

4000

-

7.67

 

Table 11: Urinalyses

Dose (ppm)

Week

Sex

VOL (ml)

Density (g/L)

PROT*Vol (mg)

PROT (g/L)

0

26

m

13.9

1023

20.3

1.78

50

26

m

14.8

1021

30.1

2.88

200

26

m

12.2

1025

56.5

5.23

1000

26

m

10.8

1032

26.7

3.29

3000

26

m

15.6

1020

31.1

3.07

0

52

m

6.3

1036

33.5

5.73

50

52

m

8.2

1035

57.9

7.95

200

52

m

7.0

1039

101.2

13.71

1000

52

m

11.2

1032

61.4

9.03

3000

52

m

9.3

1034

58.2

7.84

0

79

m

7.1

1037

35.2

5.35

50

79

m

7.8

1030

42.9

5.49

200

79

m

12.3

1023

76.1

6.53

1000

79

m

7.5

1025

36.4

5.99

3000

79

m

8.4

1028

43.5

6.37

0

103

m

11.6

1024

75.9

7.70

50

103

m

8.6

1031

77.2

8.98

200

103

m

10.9

1024

84.1

8.65

1000

103

m

8.8

1030

91.4

9.67

3000

103

m

11.8

1024

75.7

6.64

0

26

f

8.5

1026

5.5

0.68

50

26

f

14.0

1016

2.8

0.25

200

26

f

12.5

1022

2.4

0.24+

1000

26

f

6.9

1028

1.8

0.31

4000

26

f

7.3

1026

1.9

0.34

0

52

f

8.4

1029

13.3

1.25

50

52

f

9.0

1021

3.2

0.37

200

52

f

10.7

1022

4.5

0.45

1000

52

f

12.5

1015+

2.5

0.32+

4000

52

f

8.4

1030

1.8

0.29+

0

79

f

5.8

1026

10.1

1.85

50

79

f

8.0

1020+

6.3

0.91

200

79

f

11.2+

1019+

10.9

1.25

1000

79

f

10.8+

1015+

6.0

0.66

4000

79

f

10.6

1018+

3.3

0.42

0

103

f

8.4

1024

46.0

4.94

50

103

f

10.0

1021

42.5

4.80

200

103

f

7.9

1024

23.6

2.85

1000

103

f

9.8

1022

19.7

1.16+

4000 

103

 f 

7.9

1029

7.5

1.14+

+ difference against control for p<0.05

++ difference against control for p<0.01

 

Table 12: Absolute and relative organ weights (mg/100 g bw) – interim kill

Dose

Sex

Body weight

Brain

Adrenals

Heart

Liver

Spleen

Kidneys

Testes

Ovaries

Absolute organ weights

g

mg

mg

mg

mg

mg

mg

mg

mg

0

m

517

2052

54

1402

17938

853

2910

3844

-

50

m

557

2159+

53

1534

19013

944

3220

4062

-

200

m

528

2064

54

1467

17702

881

3085

3815

-

1000

m

531

2128

52

1560

19351

801

3054

3652

-

3000

m

519

2105

56

1439

21255+

715

2998

4051

-

0

f

312

1912

84

1023

10016

535

1994

-

172

50

f

302

1890

83

1031

9690

561

2057

-

173

200

f

296

1920

83

1059

9442

476

2061

-

197

1000

f

285

1879

80

988

10299

684

1983

-

204

4000

f

261++

1827

69

974

11901++

488

1856

-

162

Relative organ weights

Body weight

Brain

Adrenals

Heart

Liver

Spleen

Kidneys

Testes

Ovaries

g

mg

mg

mg

mg

mg

mg

mg

mg

0

m

517

400

10

272

3480

166

561

749

-

50

m

557

390

10

276

3424

170

580

731

-

200

m

528

393

10

279

3347