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REACH

4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-tetrazole-1-carboxamide

EC number: 605-140-1 | CAS number: 158237-07-1

Life Cycle description
Uses advised against

Toxicological information

Carcinogenicity

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Description of key information

Carcinogenicity (OECD 451), mouse:

NOAEL, neoplastic: 2000 ppm (equivalent to 575.4 and 831.1 mg/kg bw/day in males and females, respectively)

Combined Chronic Toxicity / Carcinogenicity (OECD 453), rat:

NOAEL, neoplastic: 1000 ppm (equivalent to 52.7 and 75.4 mg/kg bw/day in males and females, respectively)

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference 1

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

Reference 2

Endpoint:: carcinogenicity: oral
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 7 Nov 1994 - 26 Nov 1996
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Qualifier:: according to guideline
Guideline:: OECD Guideline 451 (Carcinogenicity Studies)
Version / remarks:: adopted 12th May 1981
Deviations:: yes
Remarks:: no differential blood count was performed in Week 52
GLP compliance:: yes
Species:: mouse
Strain:: B6C3F1
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Bomholtgard Breeding and Research Center Ltd., Denmark
- Age at study initiation: 5 - 6 weeks
- Weight at study initiation: 17 - 24 g (males), 14 - 19 g (females)
- Housing: individually in Type II Makrolon® cages on low-dust soft-wood shavings
- Diet: standard diet (Altromin®1321 meal, Altromin GmbH, Lage), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY: The nutritional composition and contaminant content of the standard diet were routinely checked and analyzed on a random basis. The tap water complied with drinking water standards in accordance with the Deutsche Trinkwasserverordnung.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- 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:: PREPARATION OF DOSING SOLUTIONS: 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).

DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with: standard diet, Altromin® 1321 containing 1% peanut oil
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: Analytical investigations on homogeneity and stability of the test compound in diet preparations were done prior to commencement of the study using samples from test mixtures (data were taken over from study T3055661). The test substance content of the food given to the animals was checked at regular intervals throughout the study (start of study, randomly each 3 month period, end of study). This was done by analyzing samples of the food mixes used. Per dose one sample of the food mixes was taken on the day the food was prepared, and another was taken after being kept under animal room conditions for the feeding period (7 days). All these samples were kept deep frozen (at temperatures of approx. -20°C) until examination.
Duration of treatment / exposure:: 24 months
Frequency of treatment:: daily per feed, ad libitum
Dose / conc.:: 20 ppm
Remarks:: corresponding to
5.4 mg/kg bw/day (males)
7.7 mg/kg bw/day (females)
Dose / conc.:: 100 ppm
Remarks:: corresponding to
28.0 mg/kg bw/day (males)
41.9 mg/kg bw/day (females)
Dose / conc.:: 500 ppm
Remarks:: corresponding to
130.6 mg/kg bw/day (males)
201.2 mg/kg bw/day (females)
Dose / conc.:: 2 000 ppm
Remarks:: corresponding to
575.4 mg/kg bw/day (males)
831.1 mg/kg bw/day (females)
No. of animals per sex per dose:: 50 (105 weeks period)
20 (control and 2000 ppm group additional satellite groups, 10 animals of each group were used for interim investigations after 27 and 53 weeks)
Control animals:: yes, plain diet
Details on study design:: - Dose selection rationale: Dose selection was based on results of a subchronic mice study (M-011023-02-1) with the following dose levels: 0, 20, 100, 600, 3600 and 7200 ppm. In the dose groups up to 100 ppm no signs of toxicity could be seen in males and females. In males given the dose 600 ppm and above body weight depression was detected. Male mice receiving 7200 ppm ingested more feed than controls. Up to the concentration of 600 ppm there were no effects on the liver. At higher doses signs of a changed liver function (increased liver weights, reduced cholinesterase activities, as well as higher cholesterol and decreased triglyceride levels in the plasma) and morphological alterations such as hypertrophic hepatocytes and single cell necroses were evident. In the dose group 600 ppm and above morphological alterations such as hypersecreting and/or hyperplastic epithelia as well as dark discolorations were found in the gallbladder, which was filled with eosinophilic concrements and/or crystals. Male mice ingesting 600, 3600 or 7200 ppm showed a reduction of cytoplasmic vacuoles in the proximal tubulus of the kidneys. However, this finding is not considered to reflect a nephrotoxic effect. In 7200 ppm females there were less vacuoles in the cells of the adrenal X-zone. In summary, there were toxic effects evident in the gall bladder and liver at 3200 and 7200 ppm, which are expected to exceed MTD criteria after long-term treatment. The morphological changes in the gall bladder and kidneys at 600 ppm were detected with a very low incidence indicating that the dietary concentration of 600 ppm is a borderline dose for these findings.

- Rationale for selecting satellite groups: Additional satellite groups receiving 0 or 2000 ppm were treated over 6 or 12 months to study occurrence in time of lesions in the gall bladder.
Observations and examinations performed and frequency:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, daily at weekends and on bank holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily, daily at weekends and on bank holidays

BODY WEIGHT: Yes
- Time schedule for examinations: before start of the study, thereafter weekly up to Week 104

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Weeks 53-54, 79 (differential blood count only) and 104-106 (bleeding for hematology and clinicochemical investigations was done separately with about a one week interval)
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes (Blood samples for the determination of glucose in deproteinized whole blood were taken from one of the caudal veins of non fasted and non-anaesthetized animals.)
- How many animals: 10 randomly selected animals from each group. As far as possible the same mice were used.
- Parameters analysed: Please refer to Table 1 under 'Any other information'.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Weeks 53-54, 79 (differential blood count only) and 104-106 bleeding for hematology and clinicochemical investigations was done separately with about a one week interval.
- Animals fasted: Yes
- How many animals: 10 randomly selected animals from each group. As far as possible the same mice were used.
- Parameters analysed: Please refer to Table 2 under 'Any other information'.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
Determination of cholinesterase (CHE) activity in the brain
- Time schedule: At necropsies in Weeks 28, 54 and at termination brain samples were taken (mice were randomly chosen) and deep frozen to be used for determination of cholinesterase activity.
Sacrifice and pathology:: GROSS PATHOLOGY: Yes (please refer to Table 3 under 'Any other information')
- Organ weights: brain, kidneys (both), liver, spleen and testes (both)

HISTOPATHOLOGY: Yes (please refer to Table 3 under 'Any other information')
Statistics:: Dunnett test in connection with a variance analysis: Body and organ weight data
Kruskal-Wallis-Test with a Steel-Test: Food intake

Descriptive Analysis: Not dichotomous varibles were described by sex, dose group and date using appropriate measures of central tendency (mean, median) and general variability (standard deviation, minimum, maximum)

Statistical Tests:
Dunnett test: used if the variables in question can be considered approximately normally distributed with equal variances across treatments
Welch test (p-value adjusted): used, if heteroscedasticity appeared more likely
ANOVA (Kruskal-Wallis test followed by adjusted Mann-Whitney-Wilcoxon tests (U-tests): If the evidence based on experience with historical data indicates that the assumptions for a parametric analysis of variance cannot be maintained, distribution-free tests were carried out.
Global tests including more than two groups are performed by sex and date, i. e. each sex x date level defines a family of tests in the context of multiple comparison procedures. Within such a family, the experiment wise error is controlled. If not otherwise noted, all pairwise tests are two-sided comparisons. Significant differences from the control group are indicated with "+" for p < 0.05 and "++" for p< 0.01.

Continuous Random Variables: Due to the right skewness often encountered in the respective empirical distributions, relative organ weights were submitted to a logarithmic transformation prior to the statistical analysis. Apart from that all variables are analyzed in the raw data form.

Discrete Random Variables: Discrete random variables with more than two possible categories are statistically evaluated using the Kruskal-Wallis test, followed by adjusted U tests.

Pathology Data: PATHDATA program, version 3.6B.

Survival Data: Survival data were not evaluated statistically

Organ weight Data: organ weights were excluded, if they differed by a factor more than 5 from the group mean
Clinical signs:: no effects observed
Description (incidence and severity):: no effects observed
Dermal irritation (if dermal study):: not examined
Mortality:: mortality observed, non-treatment-related
Description (incidence):: No treatment-related mortalities.
For details please refer to Table 4 in "any other information on results incl. tables".
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: 2000 ppm: Decreased body weight in males (9.6% main groups; 13.6% satellite group)
Food consumption and compound intake (if feeding study):: effects observed, non-treatment-related
Description (incidence and severity):: All treatment groups consumed slightly more food, both, per group and per kg body weight. No dose-dependency was apparent.
For details please refer to Table 5 in "any other information on results incl. tables".

Analsyes of test substance in the diet:
The results revealed that the test substance was homogeneously distributed in the diet amount used, and that it is stable in the concentration range used throughout the feeding period (1 week). At eleven randomly selected time points samples of all diet mixtures fed to the animals were taken and analyzed for their content of test substance and stability over the period of use. All food mixtures checked during the study proved to lie in the correct concentration range (no effects observed).
Food efficiency:: no effects observed
Description (incidence and severity):: No effects observed.
For details please refer to Table 5 in "any other information on results incl. tables".
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: not examined
Haematological findings:: no effects observed
Description (incidence and severity):: Observed significant deviations were considered not relevant since events were sporadically and dose correlations were absent.
For details please refer to Tables 6 and 7 in "any other information on results incl. tables".
Clinical biochemistry findings:: effects observed, treatment-related
Description (incidence and severity):: 500 ppm: slightly reduced glucose in females at termination; increased cholesterol in females in Week 54 and termination with no clear dose correlation; decreased triglyceride in males at termination, decreased ChE activity in erythrocytes of males in Week 54
2000 ppm: slightly reduced glucose in females at termination; increased cholesterol in females in Week 54 and termination with no clear dose correlation; decreased triglyceride in males at termination; decreased plasmatic ChE activity in females in Week 54 and at termination, decreased ChE activity in erythrocytes of males and females in Week 54
Effects observed on ChE activity in erythrocytes is considered as biologically significant, due to missing dose response and/or presence at only one time point.

Statistically significant changes in ASAT, ALAT, cholesterol (males) and urea concentration found in several groups at Week 54 were not considered related to the test substance treatment, since the deviations to control values were very slight, occurred not again at termination and were not distributed dose-dependently. The mean concentrations of creatinine, protein, albumin and bilirubin did not differ in any of the dose groups from the control values to a toxicologically relevant extent. A few significant means in these parameters were considered not relevant, since a dose dependence was not visible.
For details please refer to Tables 8 to 10 in "any other information on results incl. tables".
Urinalysis findings:: not examined
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):: 500 ppm: increased relative (females) liver weights at termination
2000 ppm: increased absolute (females) and relative (both sexes) liver weights in Weeks 28 and 54, at termination only in females (relative, dose-dependency is lacking); increased relative testes weights in Week 28, 54 and termination), increased absolute kidney and spleen weights in males at termination
For details please refer to Table 13 in "any other information on results incl. tables".
Gross pathological findings:: effects observed, treatment-related
Description (incidence and severity):: 500 ppm: dark-coloured content or stones in gallbladder of both sexes at termination
2000 ppm: dark-coloured content or stones in gallbladder of females in Week 28 and both sexes in Week 54 and at termination
For details please refer to Tables 11 and 12 in "any other information on results incl. tables".
Neuropathological findings:: not examined
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: 500 ppm:
At terminal necropsy the gallbladder contained simple or cystic mucosal hyperplasia with a distinctly increased frequency in females. The number of mice showing inspissated bile or bile concretions was enhanced.
2000 ppm:
In Week 28 the gallbladder of 7 males and all females contained eosinophilic masses. The epithelium of the gallbladder was hyperplastic (7 males, all females) and showed hypersecretory activity (9 males, all females). In some of these mice focal inflammatory infiltrations were noted in the epithelium. Hepatocytes showed cytoplasmic changes (eosinophilic and larger cells with larger nuclei) in both sexes. One female exhibited a focal necrosis and one male showed a basophilic focus in the liver.
In Week 54 the gallbladder of some males and most females contained an eosinophilic content as well as a hyperplastic, hypersecretory and/or hypertrophic epithelium accompanied with focal inflammatory infiltrations in some cases. Nearly all mice showed hepatocellular cytoplasmic changes.
At terminal necropsy the gallbladder contained simple or cystic mucosal hyperplasia with a distinctly increased frequency in both sexes. The number of mice showing inspissated bile or bile concretions was dose-dependently enhanced. Vacuoles of the proximal tubules were reduced in frequency and severity in the kidneys of males.

For details please refer to Tables 14 and 15 in "any other information on results incl. tables".
Histopathological findings: neoplastic:: no effects observed
Description (incidence and severity):: At 100, 500 and 2000 ppm there were slightly more (trend p<0.05) males exhibiting a hepatocellular adenoma (3-3-5-7-8) compared to control and 20 ppm group. These incidences did not exceed the reference range (up to 11 adenomas out of 50 males) seen in historical controls. Furthermore, there were no significant difference if adenomas and carcinomas when evaluated together (6-5-14-14-10). Therefore, a treatment-related effect was not assumed.
At 2000 ppm the frequency of uterine stromal cell sarcomas (1-1-0-0-3) was higher (trend p<0.05) than in controls. A relevant positive trend is not assumed for this tumour type, which must be classified also as common. In a two year study with B6C3F1 mice running in parallel three of 50 control females exhibited a uterine sarcoma. Additionally, three out of 50 uterine stromal cell sarcomas were seen in a mid-dose group of a (negative) oncogenicity study. Therefore, the occurrence of three uterine stromal cell sarcomas in the 2000 ppm group was not interpreted as indicative of a carcinogenic effect.
In 2000 ppm females more (trend p<0.01) mammary adenocarcinomas (0-0-0-1-3) were found than in the other groups. Since this frequency is only slightly above the reference range of studies performed at in the same laboratory (up to 1 out of 50) and within the historical data reported by the National Institute of Environmental Health Sciences, in 1991 (up to 10%) for B6C3F1 mice it was considered to be most likely incidental.
For details please refer to Tables 16 to 18 in "any other information on results incl. tables".
: Key result
Dose descriptor:: NOAEL
Remarks:: non-neoplastic
Effect level:: 100 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: 28.0 mg/kg bw/day (males) / 41.9 mg/kg bw/day (females)
: Key result
Dose descriptor:: LOAEL
Remarks:: non-neoplastic
Effect level:: 500 ppm
Based on:: test mat.
Sex:: male/female
Basis for effect level:: gross pathology; histopathology: non-neoplastic; organ weights and organ / body weight ratios
Remarks on result:: other: corresponding to: 130.6 mg/kg bw/day (males) / 201.2 mg/kg bw/day (females)
: Key result
Dose descriptor:: NOAEL
Remarks:: neoplastic
Effect level:: 2 000 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: 575.4 mg/kg bw/day (males) / 831.1 mg/kg bw/day (females)
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 500 ppm
System:: hepatobiliary
Organ:: gall bladder; liver
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: not specified
Conclusions:: CLP: STOT RE 2, H372 Liver

Endpoint conclusion

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 52.7 mg/kg bw/day
Study duration:: chronic
Species:: rat
Quality of whole database:: The available information comprises adequate and reliable studies (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.7, of Regulation (EC) No 1907/2006.
System:: urinary
Organ:: bladder

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:: no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:: no study available

Justification for classification or non-classification

The available data on the target substance for carcinogenicity do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 (CLP). Data are therefore conclusive but not sufficient for classification.

Additional information

In rats, urothelial hyperplasia and transitional cell tumours of the urinary bladder were observed in a 13-week and a 2-year carcinogenicity study (1997u, 1997f). In detail, in a 13-week study in rats, urothelial hyperplasia of the urinary bladder was observed at the highest dose of 6400 ppm, which was reversible within a period of 4 weeks (1997u). In the 2-year carcinogenicity study in rats, urothelial hyperplasia was observed at the highest dose, which was 3000 ppm in males and 4000 ppm in females (1997f). In females dosed at 4000 ppm also transitional cell tumours of the urinary bladder were present.

Based on additional electron microscopical investigations, observed changes in the urinary bladder were considered to represent mild regenerative urothelial hyperplasia, in consequence of cytotoxicity (1997u). This was supported by published literature [1, 2, 3], demonstrating that the metabolites of the test substance cyclo-hexylamine (CA) and N-ethylcyclohexylamine (CEA) are corrosive and cytotoxic and are therefore most likely responsible for the induction of regenerative hyperplasia in the urinary bladder. Therefore single transitional cell tumours in female high-dose rats were considered the consequence of chronic irritation and resulting regenerative proliferation and not a sign of a primary carcinogenicity.

This is supported by numerous negative in-vitro and in-vivo studies on genetic toxicity (1995e, 1995f, 1996e, 1996f, 1997l, 1996g, 1995g, 1997m) which exclude a genotoxic mechanism. Of note is a specific in-vivo study in rats that demonstrated the absence of DNA-adducts in the urinary bladder after exposure to the test substance (1997m). In addition, genetic toxicity studies with the metabolites chlorphenyltetrazolinone (CPT), 4-hydroxy CEA, CA and CEA did not indicate a genotoxic potential (2000e).

In summary, a non-genotoxic, epigenetic mechanism for the tumour development is suggested for the test substance for which a threshold can be determined. Since urinary bladder tumours were only observed at the highest dose of 4000 ppm in females, 1000 ppm was determined as NOEL for tumour development, corresponding to 75 mg/kg bw/day. This high dose effect was confirmed in a 13-week metabolism study in rats (1998a). Here it was shown that exposure at a high-dose (6400 ppm) leads to exhaustion of the glutathione detoxification capacity which is well functioning at a dose of 50 ppm, leading to a qualitative and quantitative altered detoxification metabolism. Hence, observed effect on the urinary bladder in rats is a high dose phenomenon and relativizes the occurrence of tumours for risk assessment in the human, for which an exposure only at a lower dose and also for a shorter period is assumed. It is therefore concluded that taking into account proper handling, a carcinogenic effect on the human can be excluded for both production and handling of the test substance as well as for the consumption of food containing residues of the test substance.

In the sub-chronic (1997h) and two-year studies (1997n) in mice, a simple or cystic (with hypersecretion of mucinous substances) mucosa hyperplasia of the gallbladder epithelium was observed in high doses. The lowest NOAEL for these findings was 100 ppm. Proliferating cell nuclear antigen (PCNA)-investigations revealed a statistically significantly increased labelling index, indicating increased cell proliferation, only at 7200 ppm. (2000b). Gallbladder tumours were not observed (2000d). Gallbladders contained darkly discoloured bile and eosinophilic amorphous substances. Increased liver weights and histological findings indicated microsomal liver enzyme induction. In a special mechanistic study, changed bile contents were identified to consist mainly of taurocholic acid, indicating an increased bile acid synthesis, obviously due to the strong induction of hepatic cytochrome P-450-dependent monooxygenases (Phase I enzymes) (1997i). No significant amounts of the test substance and of the main metabolite o-chlorophenyltetrazolinone (CPT) were found in the bile.

In sub-chronic (1996c) and chronic (1997e) studies with the test substance in Beagle dogs a dose-dependent microsomal (Phase I) enzyme induction and marginal to slight liver damage at higher dose levels was observed. In addition, gallbladder hyperplasia was observed at high dietary concentrations. The NOAEL for gallbladder mucosa hyperplasia were 75 ppm, equal to 2.95 mg/kg bw/day (females from sub-chronic study) and 40 ppm, equal to 1.12 mg/kg bw/day (males from chronic study). In contrast to the finding in mice, additional characterization of gallbladder effects revealed the absence of any statistically significantly increased PCNA-labeling index, indicating no increased cell proliferation, at all tested doses (75, 300 and 1200 ppm) in dogs (2000b).

The reason of gallbladder effects in dogs were further investigated in special mechanistic studies (1997g, 1998d, 2000a). In these studies, female dogs that received 750 ppm test substance (the highest dietary concentration used in the chronic study) for 6 weeks, showed the before observed effects on liver and gallbladder and an increased activity of hepatic cholesterol 7 alpha hydroxylase (CYP7A).

In addition, changes in biliary flow and acid production were observed in a special study in bile duct cannulated dogs. In this study dogs were again treated at 750 ppm test substance for 6 weeks, but the gallbladder was surgically separated from the cannulated bile stream. The bile duct-cannulated dogs showed no gallbladder hyperplasia, indicating that the hyperplasia observed in previous studies is not a systemic, but a local effect, caused by the altered bile. It was assumed that the increased CYP7A activity led to an increased bile acid biosynthesis which possibly partly exhausted the taurine conjugation capacity of the liver. The resulting altered bile composition with an increased level of free bile acids (known to have a higher irritating potential than conjugated bile acids) was considered to be the basis for the gallbladder mucosa hyperplasia observed in previous studies. This is also supported by data showing only low concentrations of the test substance and its metabolites in the bile, making a direct test substance-related effect unlikely.

In the rat, no gallbladder effects were observed. Special mechanistic studies in the rat revealed a different pattern of liver enzyme induction after treatment with the test substance (1997j). In rats, the test substance was a strong inducer of Phase II enzymes, but not of Phase I enzymes (including CYP7A) as observed in mice and dogs. In addition, changes in bile acid content were not observed in treated rats (2000c). Furthermore, in the bile fluid of rats, no test substance and only low concentrations of the metabolites CPT and cyclohexyl-o-glucuronide were detected.

Genotoxicity studies with the test substance, a study with the bile from mice treated with the test substance and studies with the following rat metabolites CPT, cyclohexylamine (CA), N-ethylcyclohexylamine (CEA) and 4-hydroxy CEA, did not show a genotoxic potential of the test substance and its metabolites (2000e, no further source available). Thus, a genotoxic pathogenesis for the gallbladder epithelium hyperplasia observed in mice and dogs was excluded.

Based on sub-chronic, chronic and mechanistic studies in mice and dogs and taking into account genotoxicity data the following was concluded:

The test substance induces mild to marked hyperplasia of the gallbladder epithelium but no gallbladder tumours in higher doses in mice and dogs.

A genotoxic pathogenesis for the gallbladder hyperplasia observed in mice and dogs was excluded, based on numerous negative genotoxicity studies with the test substance and its metabolites.

A primary local effect of the test substance on the gallbladder mucosa was considered very unlikely, since in mice no significant and in dogs only low amounts of the test substance and its main metabolite CPT were found in the bile, indicating no relevant biliary excretion of the test substance.

A systemic effect of the test substance or its metabolites was also not assumed, since a mechanistic study with bile duct cannulated dogs demonstrated the absence of gallbladder effects, when the gallbladder was surgically separated from the bile stream. Taken together no genotoxic, no primary local and no systemic promoting effect of the test substance and its metabolites is assumed for the pathogenesis of gallbladder hyperplasia.

Based on several mechanistic studies an indirect mechanism for gallbladder epithelium hyperplasia is assumed. It was demonstrated that the test substance causes microsomal (Phase I) liver enzyme induction in mice and dogs, including CYP7A, which is the first and rate limiting enzyme in the conversion of cholesterol to bile acids. It was concluded that this enzyme induction results in an altered bile flow and bile composition. This altered bile composition with increased levels of (free) bile acids was considered the pathogenic basis for the observed gallbladder mucosa hyperplasia in mice and dogs.

For more details please refer to the endpoint summary of Chapter 7.5 “Repeated dose toxicity”.

References:

[1] MDL Information Systems, Inc., 14600 Catalina Street, San Leandro CA 94577, USA, 1996, Substance: Cyclohexylamine, Section 11 - Toxicological Information

[2] MDL Information Systems, Inc., 14600 Catalina Street, San Leandro CA 94577, USA, 1996, Substance: N-Ethylcyclohexylamine, Section 11 – Toxicological Information

[3] Cattanach, B. M., 1976 The Mutagenicity of Cyclamates and their Metabolites, Mut. Res. 39(1), 1-28

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

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

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

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

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

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

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

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

	pH mean value
Sex	male	female
Dose (ppm)	male	female
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

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+

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
Relative organ weights		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

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

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

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

Dose (ppm)	0	50	100	500	2000
sex (male)
Week 26	0	1	0	0	0
Week 51	0	2	0	0	0
Week 78	2	2	0	1	1
Week 104	4	3	3	4	3
sex (female)
Week 26	1	0	0	0	0
Week 51	1	2	0	0	0
Week 78	2	2	3	1	4
Week 104	11	16	16	10	17

Intake food
			g/animal		g/kg body weight
Dose (ppm)	Sex	Days	Total	per Day	Total	per Day
0	m	707	4760	6.7	172106	243.4
50	m	707	5203	7.4	192396	272.1
100	m	707	5305	7.5	197896	279.9
500	m	707	5093	7.2	184713	261.3
2000	m	707	5420	7.7	203414	287.7
0	f	707	6062	8.6	256803	363.2
50	f	707	6310	8.9	272780	385.8
100	f	707	6799	9.6	296411	419.3
500	f	707	6666	9.4	284511	402.4
2000	f	707	6896	9.8	293796	415.6
Intake of test compound
			mg/animal		mg/kg body weight
Dose (ppm)	Sex	Days	Total	per Day	Total	per Day
50	m	707	104	0.1	3848	5.4
100	m	707	531	0.8	19790	28.0
500	m	707	2546	3.6	92356	130.6
2000	m	707	10839	15.3	406827	575.4
50	f	707	126	0.2	5456	7.7
100	f	707	680	1.0	29641	41.9
500	f	707	3333	4.7	142255	201.2
2000	f	707	13793	19.5	587593	831.1

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
Relative organ weights		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

Dose			LEUCO	ERY	HB	HCT	MCV	MCH	MCHC	THRO
ppm	sex	week	10⁹/L	10¹⁹/L	g/L	L/L	fl	pg	g/LERY	10⁹/L
0	m	53	5.1	9.08	142	0.421	46.4	15.6	336	1145
20	m	53	5.3	9.12	144	0.421	46.1	15.8	342	1052+
100	m	53	5.6	9.14	143	0.420	45.9	15.6	340	1104
500	m	53	5.7	9.28	145	0.427	46.0	15.7	341	1146
2000	m	53	6.1	9.15	141	0.423	46.2	15.4	334	1099
0	m	104	8.1	8.39	137	0.405	49.0	16.4	336	1195
20	m	104	5.5+	8.80	138	0.420	47.7	15.7	329	1379
100	m	104	6.7	8.71	135	0.407	47.0	15.6	332	1475+
500	m	104	6.2	9.13	143	0.430	47.0	15.7	335	1410
2000	m	104	6.4	8.73	139	0.411	47.1	15.9	338	1479+
0	f	53	4.1	9.34	144	0.430	46.0	15.4	335	910
20	f	53	4.4	9.44	145	0.437	46.3	15.4	332	911
100	f	53	3.8	9.31	143	0.427	45.9	15.4	334	946
500	f	53	4.3	9.33	144	0.430	46.1	15.5	336	941
2000	f	53	4.2	9.24	143	0.428	46.3	15.5	335	941
0	f	104	9.7	8.17	132	0.392	47.9	16.1	337	620
20	f	104	2.7	8.48	134	0.400	47.2	15.8	334	931 +
100	f	104	3.1	8.26	132	0.398	48.1	15.9	331	847
500	f	104	3.9	8.44	132	0.399	47.3	15.6	331	798
2000	f	104	3.1	8.41	135	0.403	47.9	16.1	336	873

Dose	Week	Sex	ASAT (GOT)	ALAT (GPT)	APh
ppm			U/L	U/L	U/L
0	54	m	34.9	26.5	90
20	54	m	32.0	25.0	88
100	54	m	30.8	25.8	93
500	54	m	42.3	44.3	99
2000	54	m	33.9	21.5+	98
0	105/106	m	31.4	34.4	88
20	105/106	m	39.2	43.4	87
100	105/106	m	49.7	63.1	105
500	105/106	m	39.4	37.9	115
2000	105/106	m	34.5	30.5	104
0	54	f	31.1	20.6	177
20	54	f	38.2+	33.7	155
100	54	f	38.5+	33.7++	159
500	54	f	36.4+	27.5++	152
2000	54	f	36.9+	33.2+	164
0	105	f	41.7	28.6	432
20	105	f	58.3	46.5	392
100	105	f	52.8	33.5	367
500	105	f	46.6	34.6	363
2000	105	f	44.2	42.9	477

Dose	Week	Sex	GLUCOSE	CHOL	CREA	UREA	BILI-t	PROT	ALB	TRIGL
ppm			mmol/L	mmol/L	µmol/L	mmol/L	µmol/L	g/L	g/L	mmol/L
0	54	m	5.88	3.06	25	13.05	1.6	56.3	29.0	2.28
20	54	m	6.05	3.46+	24	13.58	1.7	57.5	29.5	2.70
100	54	m	6.64++	3.60++	26	14.16	1.8	59.2+	30.3	2.93
500	54	m	6.17	3.73++	25	13.44	1.8	59.4+	30.2	2.55
2000	54	m	6.09	3.52+	27	12.91	1.8	57.2	29.9	1.88
0	105/106	m	6.11	3.75	26	10.60	1.3	58.6	28.0	2.45
20	105/106	m	6.11	3.93	27	11.00	1.4	59.9	28.8	1.81
100	105/106	m	6.14	4.36	27	11.02	1.5	63.3	28.8	1.50+
500	105/106	m	5.99	3.91	24	10.51	1.6	58.7	26.8	1.28+
2000	105/106	m	6.38	4.67	26	10.16	1.5	61.8	29.7	1.19++
0	54	f	6.50	2.46	26	11.34	1.5	54.9	30.3	1.30
20	54	f	6.44	2.51	25	11.42	1.6	55.9	30.5	1.14
100	54	f	6.55	2.55	27	13.31++	1.4	55.7	30.2	1.42
500	54	f	7.10	2.93++	25	13.37++	1.4	56.2	29.7	1.27
2000	54	f	6.78	2.97++	26	13.07+	1.4	57.0	29.5	1.13
0	105	f	6.37	2.09	26	11.54	1.6	56.4	29.1	1.23
20	105	f	5.96	3.38	27	11.95	1.8	61.1	30.7	1.30
100	105	f	6.13	2.96	27	12.00	1.8	58.9	30.4	1.20
500	105	f	5.58++	2.98+	26	12.32	1.7	59.0	29.2	1.19
2000	105	f	5.53++	2.92++	26	11.03	1.5	60.8	29.6	1.02

	Necropsy Week 28				Necropsy Week 54
Sex	male		female		male		female
Dose ppm	0	2000	0	2000	0	2000	0	2000
Change in contents				1		2		8
Dilatation/s				1				7
Discoloration/s				8

Registration Dossier

Registration Dossier

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Diss Factsheets

4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-tetrazole-1-carboxamide

Carcinogenicity

Administrative data

Description of key information

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

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Reference 1

Reference 2

Endpoint conclusion

Carcinogenicity: via inhalation route

Endpoint conclusion

Carcinogenicity: via dermal route

Endpoint conclusion

Justification for classification or non-classification

Additional information

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