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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

Endpoint summary

Administrative data

Description of key information

Chronic Toxicity (OECD 452), oral, dog:

NOAEL, non-neoplastic: 20 ppm (equivalent to 0.55 and 0.52 mg/kg bw/day in males and females, respectively)

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

NOAEL, non-neoplastic: 200 ppm (equivalent to 10.3 and 14.6 mg/kg bw/day in males and females, respectively)

Subacute Toxicity (OECD 410), dermal, rat:

NOAEL, systemic: 250 mg/kg bw/day

NOAEL, local: 1000 mg/kg bw/day (7.5 mg/cm²)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference 1

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

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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 55% ± 5%
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:: oral: feed
Vehicle:: unchanged (no vehicle)
Details on oral exposure:: - DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- The test substance was blended (using a mixing granulator manufactured by Loedige, Paderborn) with Altromin® 1321 containing 1% peanut oil to minimize dust formation (including 0 ppm concentration).
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: Analytical investigations to demonstrate homogeneity and stability of the test compound in diet preparations were done prior to commencement of the study. The test substance content of diet mixtures fed to the animals was checked analytically at regular intervals throughout the study (start of study, randomly each 3 month period, end of study). Per dose one sample of the food mixtures was taken on the day the mixture was prepared, and another was taken after being kept underanimal room conditions for the feeding period (7 days). All these samples were kept deep frozen (at temperatures of approx. -20°C) until analysis.
Duration of treatment / exposure:: 24 months
Frequency of treatment:: daily per feed, ad libitum
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 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
Effect level:: 200 ppm
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: no adverse effects observed up to this dose level
Remarks on result:: other: corresponding to: 10.3 mg/kg bw/day (males) / 14.6 mg/kg bw/day (females)
: Key result
Dose descriptor:: LOAEL
Effect level:: 1 000 ppm
Based on:: test mat.
Sex:: male/female
Basis for effect level:: histopathology: non-neoplastic; other: Cholinesterase activity in erythrocytes and brain
Remarks on result:: other: corresponding to: 52.7 mg/kg bw/day (males) / 75.4 mg/kg bw/day (females)
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 1 000 ppm
System:: hepatobiliary
Organ:: liver
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: not specified
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 1 000 ppm
System:: nervous system
Organ:: other: cholinesterase activity inhibited in erythrocytes (1000 ppm and above) and brain (4000 ppm) (statistically significant inhibition by 20% or more in erythrocytes or brain is considered a clear toxicological effect)
Treatment related:: yes
Dose response relationship:: yes
Conclusions:: CLP: STOT RE 2, H372 Liver

Reference 2

Endpoint:: chronic toxicity: oral
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 4 Apr 1996 - 7 Apr 1997
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Qualifier:: according to guideline
Guideline:: OECD Guideline 452 (Chronic Toxicity Studies)
Version / remarks:: adopted May 1981
Deviations:: no
GLP compliance:: yes
Species:: dog
Strain:: Beagle
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: White Eagle Laboratories, Doylestown, Pennsylvania
- Females nulliparous and non-pregnant: yes
- Age at study initiation: not greater than six month of age
- Weight at study initiation: 4.7 - 9.6 kg (males), 5.2 - 8.9 kg (females)
- Housing: individually housed in stainless steel runs
- Diet: Purina Mills Lab Canine Diet 5006-3, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 6 days

DETAILS OF FOOD AND WATER QUALITY: Feed (food) and corn oil were periodically sampled and analyzed for potential contaminants by PMI Feeds, Inc. and Hazleton Wisconsin labs, respectively. Tap water (Kansas City Municipal Water) was analyzed for impurities by Kansas City Water Department and Pace Incorporated.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 28.9
- Humidity (%): 30 -70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 25 Mar 1996 To: 9 Apr 1997
Route of administration:: oral: feed
Vehicle:: corn oil
Details on oral exposure:: - PREPARATION OF DOSING SOLUTIONS: the test substance was mixed in the feed at the designated nominal concentrations on the basis of the active
ingredient

- DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with: Purina Mills Lab Canine Diet 5006-3
- Storage temperature of food: freezer conditions

- VEHICLE
- Justification for use and choice of vehicle: corn oil 1% weight of the the diet
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: Analysis of the test substance in the diet:
Test substance concentration in ration was determined by liquid chromatographic (LC) analysis. Recovery samples were analyzed with each group of test concentration determinations. The mean percent recovery, standard deviation (SD) and the coefficient of variation (CV) were determined for the recoveries.

Homogeneity in the food:
Homogeneity and stability studies found the distribution of test substance mixed with dog ration was determined for three concentrations. Dog ration was prepared with 20, 50 and 2000 ppm test substance. Three samples were taken from three areas of a mixing bowl designated as top, middle and bottom for a total of nine samples. The mean, SD and CV were determined for samples analyzed from each concentration.

Stability in the food:
Homogeneity and stability studies of test substance mixed with dog ration was assessed under freezer conditions (approximately -23°C) for 28 days and at room temperature (approximately 22°C) for 14 days at three concentrations. Test substance mixed with 20, 50 and 2000 ppm was sampled using the following procedure. After the mixing procedure was completed, an initial sample was taken immediately from each level for analysis. In order to simulate actual study conditions, the ration was placed in the freezer for seven days prior to the room temperature stability test. Prior to the placement of the ration into the freezer, it was divided into two portions.
After seven days in the freezer, one portion was removed for room temperature testing. The room temperature portion was sampled for analysis on Days 0(7), 1(8), 3(10), 7(14), 10(17) and 14(21). The parenthetical numbers are the actual study days. The portion remaining in the freezer was sampled for analysis on Days 7, 14, 21 and 28. The 20 ppm concentration level freezer storage stability was also investigated for 56 days.

Verification of test compound in the food:
A minimum of two batches of treated ration per level were prepared weekly for the study and stored in the freezer until required. Samples of ration were taken from each prepared batch. Samples for concentration analysis were a composite of all batches mixed for a treatment level. The concentration was verified for the first three weeks of the study to assure the diet was being properly prepared and at random intervals throughout the study. The mean, SD and CV of the test substance concentrations were determined for each test level.
Duration of treatment / exposure:: 12 months
Frequency of treatment:: daily
Dose / conc.:: 20 ppm
Remarks:: corresponding to
0.55 mg/kg bw/day (males)
0.52 mg/kg bw/day (females)
Dose / conc.:: 40 ppm
Remarks:: corresponding to
1.12 mg/kg bw/day (males)
1.14 mg/kg bw/day (females)
Dose / conc.:: 200 ppm
Remarks:: corresponding to
5.35 mg/kg bw/day (males)
5.50 mg/kg bw/day (females)
Dose / conc.:: 750 ppm
Remarks:: corresponding to
24.32 mg/kg bw/day (males)
24.74 mg/kg bw/day (females)
No. of animals per sex per dose:: 4
Control animals:: yes, concurrent vehicle
Details on study design:: - Dose selection rationale: The nominal concentrations chosen for this study were 0 (concurrent vehicle control) 20, 40, 200 and 750 ppm of technical grade test substance to be mixed in the feed on the basis of the active ingredient. Selection of these concentrations was based on a subchronic feeding study with doses of 75, 300 and 1,200 ppm in the diet, where the liver appeared to be a target organ. At 75 ppm, N- and O-demethylase induction occurred but without microscopic hepatic changes and was considered to be a no-observable-adverse-effect level (NOAEL). Also during this subchronic study, serum hepatic enzymes (ALT and ALP) were elevated at 300 ppm and higher, and food consumption and body weights were reduced at 1,200 ppm.
Observations and examinations performed and frequency:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- The following cage side observations were checked: mortality, morbidity, the ability of the animal to access feed and water and the presence of feed wastage

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly and immediately prior to necropsy

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: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: following the period of quarantine/acclimation and prior to initiation of dosing, after 6 months of dosing and prior to termination of the study
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: pre-treatment and approximately 3, 6, 9 and 12 (study termination) months after initiation of dosing
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters analysed: morphologies, erythrocytes, Heinz bodies, haemoglobin, haematocrit, leucocytes (total & differential), mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, mean corpuscular volume, platelets, reticulocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: pre-treatment and approximately 3, 6, 9 and 12 (study termination) months after initiation of dosing
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters analysed: Alanine Aminotransferase (ALT), Albumin (Alb), Alkaline Phosphatase (ALP), Aspartate Aminotransferase (AST), Total Bilirubin (T-Bili), Bile Acids, Blood Urea Nitrogen (BUN), Calcium (Calc), Chloride (Cl), Cholinesterase Plasma (PChe)/Erythrocyte (RChe)/ Brain (BChe), Total Cholesterol (Chol), Creatinine (Creat), Creatine Kinase (CK), Cytochrome P-450 (Cytop450), Gamma-Glutamyl Transpeptidase (GGT), Globulin (Glob), Glucose (Gluc), Lactic Dehydrogenase (LD), Phosphorus (Phos), Potassium (K), Total Protein (T-Prot), N-Demethylase (N-Demeth), O-Demethylase (O-Demeth), Sodium (Na), Triglycerides (Trig), Uric Acid (Uric-A), Triiodothyronin (T3), Thyroxine (T4), Thyroid Stimulating Hormone (TSH)

URINALYSIS: Yes
- Time schedule for collection of urine: pre-treatment and approximately 3, 6, 9 and 12 (study termination) months after initiation of dosing
- Metabolism cages used for collection of urine: No
- Animals fasted: No
- Parameters analysed: Appearance, Specific gravity, Protein (PRO), Ketones (KET), Urobilinogen (URO), Nitrites (NIT), Microscopic examination of sediment, pH, Glucose (GLU), Occult blood (BLD), Bilirubin (BIL), Leukocytes (LEU)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: following the period of quarantine/acclimation and prior to initiation of dosing and prior to termination of the study
- Dose groups that were examined: all
- Battery of functions tested: mental status/behaviour, gait characteristics, postural status and reactions, spinal/cranial reflex tests, In addition, thoracic auscultation of the heart and lungs, and rectal body temperature

OTHER:
Electrocardiogram/Blood Pressure Assessments
- Time schedule for examinations: pre-treatment and prior to termination
- How many animals: all
- Lead II measurements of the P wave, QRS complex and T wave, typical of that animal were taken. Non-invasive blood pressure (BP) measurements of systolic, diastolic, mean arterial pressure and heart rate were taken, once prior to administration of the test substance and just prior to study termination
Sacrifice and pathology:: GROSS PATHOLOGY: Yes
- The following tisues were collected: adrenals, aorta, bone, femur, bone, rib/cc jct, bone, sternum, bone marrow, brain** (cerebellum, cerebrum-midbrain, medulla/pons), cecum, cervix, colom, ears, internal structures, epididymis, esophagus, eyes, oviducts (fallopian tubes), gall bladder, gross lesions, heart, joint, fem/tib, kidneys, larynx, liver*, lungs, lymph node (mesenteric, retropharyngeal), mammary gland, muscle protocol, nasal structures, nasopharync, nerve optic, nerve sciatic, ovaries, pancreas, parathyroid, physical identifier, pituitary, prostate, rectum, salivary glands, akin, protocol, small intestine (duodenum ileum, jejunum), spinal cord (cervical, lumbar, thoracic), spleen, stomach, testicles, third eyelid/lacrimal gland, thymus, thyroid with parathyroids, trachea, urinary bladder, uterus
* Portions of liver tissue (at least 3 sections) were used for enzyme analyses.
** Portion of the brain was taken for cholinesterase.
- Organ weights: brain, heart, liver, lungs, spleen, kidneys, pituitary, adrenal glands, testicles, thymus, ovaries, thyroid (with parathyroids)

HISTOPATHOLOGY: Yes, all tissues except internal ear structures, and nasal structures
Statistics:: ANOVA (analysis of variance to determine if statistical differences existed among the groups tested)

Student's t-test (comparison of treatment grpup to control group, confidence intervals at 95% were utilized to assess biological significance. Additional statistical tests for hypothesis testing were conducted at the study director's discretion. A probability value of p<0.05 was accepted as significant.)

Chi-Square procedure ( Pathology frequency data, incidences differ statistically among the groups tested.)
If significance was suggested, each treatment group was compared to the control using a Fisher's Exact test. A probability value of p<0.05 was accepted as significant for all statistical tests, with the exception of Bartlett's test, in which p<0.001 was used.

SAS software (Statistical analysis of micropathology data.)

DATATOX software (all other statistical evaluations)

Chi-Square procedure followed by a one-tailed Fisher's Exact Test in cases of significant variation by the Chi-Square analysis (necropsy and micropathology frequency data)

Analysis of Variance (ANOVA), followed by Student's t test, on parameters showing a significant effect by the initial ANOVA (terminal body weight, absolute and relative organ weights, and clinical pathology data) Use of the term "significant" in this report refers to stalls tical significance, unless designated otherwise.
Clinical signs:: effects observed, non-treatment-related
Description (incidence and severity):: There were no relevant clinical signs or observations that were attributed to chronic test substance administration in the diet. For details please refer to the pdf "M-011537-01-1_Chronic Toxicity Feeding Study in the Beagle Dog selected tables" under attached background material.
Mortality:: mortality observed, non-treatment-related
Description (incidence):: One mortality was observed at Day 146 due to progressive emaciation and deteriorating body condition. Gross necropsy revealed pyloric stenosis and dilated cerebral ventricles that were most likely congenital abnormalities that could not be detected clinically at the pre-treatment physical examination. There was also a reduction in the size of the thymus. Microscopically chronic enteritis was observed.
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: 750 ppm: decreased body weights in both sexes (-8.5% /-21.7% in males/females at study termination)
For details please refer to the pdf "M-011537-01-1_Chronic Toxicity Feeding Study in the Beagle Dog selected tables" under attached background material.
Food consumption and compound intake (if feeding study):: effects observed, treatment-related
Description (incidence and severity):: 750 ppm: increased food consumption in both sexes
For details please refer to the pdf "M-011537-01-1_Chronic Toxicity Feeding Study in the Beagle Dog selected tables" under attached background material.
Food efficiency:: effects observed, treatment-related
Description (incidence and severity):: 750 ppm: reduced body weight gain in both sexes
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: no effects observed
Description (incidence and severity):: no effects observed
Haematological findings:: effects observed, treatment-related
Description (incidence and severity):: 200 ppm: relative neutrophil (SEG): lymphocyte (LYM) ratio reversal in males
750 ppm: increased platelet counts in both sexes, relative neutrophil (SEG): lymphocyte (LYM) ratio reversal in males
For details please refer to the pdf "M-011537-01-1_Chronic Toxicity Feeding Study in the Beagle Dog selected tables" under attached background material.
Clinical biochemistry findings:: effects observed, treatment-related
Description (incidence and severity):: 20 ppm: increased N- and O-demethylase in both sexes (but within or at the upper limit of the historical control range)
40 ppm: increased N- and O-demethylase in both sexes, increased cyp P-450, ALP in females
200 ppm: increased N- and O-demethylase, cyp P-450 and ALP in both sexes, increased ALT, GGT in females, decreased albumin and total protein in females
750 ppm: increased N- and O-demethylase, cyp P-450, ALP, ALT and GGT in both sexes, decreased albumin and total protein in both sexes, decreased calcium in females, increased T3 and T4 in males
For details please refer to Table 4 in "any other information on results incl. tables" and the pdf "M-011537-01-1_Chronic Toxicity Feeding Study in the Beagle Dog selected tables" under attached background material.
Urinalysis findings:: no effects observed
Description (incidence and severity):: no effects observed
Behaviour (functional findings):: no effects observed
Description (incidence and severity):: no effects observed
Immunological findings:: not examined
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Description (incidence and severity):: 40 ppm: non-significant tendency toward increased absolute liver weights in females
200 ppm: non-significant tendency toward increased absolute liver weights in both sexes, increased thyroid weights in females (absolute and relative)
750 ppm: increased liver weights in males (absolute and relative) and females (relative); absolute weights for females were increased but not statistically significant, increased thyroid weights in males (relative) and females (absolute and relative), increased relative adrenal weights in males
For details please refer to Table 1 in "any other information on results incl. tables" and the pdf "M-011537-01-1_Chronic Toxicity Feeding Study in the Beagle Dog selected tables" under attached background material.
Gross pathological findings:: effects observed, treatment-related
Description (incidence and severity):: 750 ppm: thickening of the gall bladder wall in one male, enlargement of the liver in one animal of each sex
For details please refer to Table 2 in "any other information on results incl. tables" and the pdf "M-011537-01-1_Chronic Toxicity Feeding Study in the Beagle Dog selected tables" under attached background material.
Neuropathological findings:: no effects observed
Description (incidence and severity):: no effects observed
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: 200 ppm: hyperplasia of the gall bladder mucosa in males, hepatocytomegaly in both sexes
750 ppm: hyperplasia of the gall bladder mucosa, hepatocytomegaly and increased frequency and/or severity of vacuolation in the inner adrenal zona fasciculata and in the zona reticularis in both sexes
For details please refer to Table 3 in "any other information on results incl. tables" and the pdf "M-011537-01-1_Chronic Toxicity Feeding Study in the Beagle Dog selected tables" under attached background material.
Histopathological findings: neoplastic:: no effects observed
Description (incidence and severity):: no effects observed
Other effects:: no effects observed
Description (incidence and severity):: no effects observed
Details on results:: Haematological findings:
- Platelet counts (PLT) were significantly increased at several intervals (including pretreatment values) in all males groups. Female PLT was increased in the 750 ppm group only. Only the change at 750 ppm is believed to be treatment-related, as all other groups followed the normal pattern (of the control) of a gradual decrease over time, while the 750 ppm group increased over time.
- A relative neutrophil (SEG): lymphocyte (LYM) ratio reversal was present in 200 and 750 ppm males at several intervals, as manifest by significant decreases in SEG percentage and increases in LYM percentages. Examination of absolute neutrophil and lymphocyte numbers indicated no statistical differences between groups at any interval; therefore, the changes are thought to be due to normal variation and not biologically significant.

Clinical biochemistry findings:
- Phase I inducible hepatic enzymes measured in the liver at study termination were increased over control levels. N- and O-demethylases were significantly increased in all treated groups (both sexes), however the 20 ppm level (both sexes) were within or at the upper limit of the historical control range. No histopathological changes were seen at 20 or 40 ppm, supporting the conclusion that this degree of Phase I induction was not an adverse effect, rather an expected physiological response to detoxification. The smallest increase was 175% of the mean control level. Cytochrome P-450 levels were significantly increased in the 200 and 750 ppm groups, both sexes, and in 40 ppm females.
- Alanine aminotransferase (ALT) was significantly increased at several intervals throughout the study (particularly toward the end) in 750 ppm groups of both sexes and in 200 ppm females.
- Alkaline phosphatase (ALP) was significantly increased in 200 and 750 ppm groups of both sexes and in 40 ppm females at six months and later.
- Gamma-glutamyl transpeptidase (GGT) was significantly increased over control levels in 750 ppm dogs of both sexes at most intervals. The 200 ppm female value was similarly increased at six months only. One decrease in GGT from control levels was disregarded as a statistical aberration.
- Albumin and total protein were significantly decreased in 750 ppm dogs of both sexes at most intervals during compound administration. Significant decreases in albumin were also noted in 200 ppm females at all
intervals including pretreatment but in total protein at nine months only. The decrease in albumin in the 750 ppm group appears to account for the less prominent changes in total protein and appears treatment-related in that group. This is not the case in the 200 ppm group. Due to the low baseline relative to control, significant pretreatment differences, and lack of temporal effect, the change was not considered biologically significant in the 200 ppm group.
- Significantly decreased calcium (CA) was noted in 750 ppm females at 3, 9, and 12 months and in 200 ppm females at three months. There was no clinical effect noted. This change is likely secondary to the decreased serum albumin noted above, as hypocalcemia is known to occur in hypoalbuminemic dogs.
- Other changes: Triiodothyronine (T3) and thyroxine (T4) levels were significantly increased in 750 ppm males at 12 months only. Thyroid stimulating hormone (TSH) levels were significantly increased in 200 ppm males at nine months only. No significant changes in thyroid parameters were noted in females. There was also a non significant tendency for T3 and T4 values to increase slightly in the 750 ppm group of both sexes during treatment. The biological significance of these changes and the significant changes noted in males was equivocal due to the wide normal variation in thyroid hormone levels and the somewhat higher (than control) levels in the pretreatment males. Several potassium (K) values were elevated over control values in males. These values were scattered in time (including some pretreatment values) and had no dose-relationship; therefore, they were considered to be statistical aberrations.

Organ weight findings including organ / body weight ratios:
- Hepatic weights were significantly increased over concurrent control values in 750 ppm males (absolute and relative weights) and females (relative weights). Absolute weights for females were increased but were not statistically significant. Overall, a non-significant tendency toward increased absolute weights was also noted in 200 ppm dogs of both sexes and in 40 ppm females. (Male 200 ppm absolute and female 40 ppm relative weights were also significantly increased.
- Thyroid relative weights were significantly increased over control in 750 ppm groups of both sexes. Absolute and relative weights were significantly increased in 200 ppm and the 750 ppm (absolute only) females. There was a generalized increase in both absolute and relative weights in both sexes at 200 and 750 ppm, but values other than those mentioned above were not significantly changed.
- Adrenals at 750 ppm of both sexes were slightly heavier than in other groups, but only the values for male relative weight were statistically significant.

Gross pathological findings:
Thickening of the gall bladder wall was noted in one 750 ppm male. Enlargement of the liver was noted in one 750 ppm dog of each sex. Other gross changes were noted without relation to test substance administration: pituitary cysts -coded as sex-incidence per group in order of increasing dose levels- (M-0, 1, 0, 0, 0; F-0, 0, 0, 1, 1) raised/discolored zones on the spleen (M-l, 0, 0, 1,0; F-0,0,0, 1, 0), thymus reduced in size (M-l, 0, 0, 1,0; F-0, 0,0, 0, 1), and dilated ventricles in the brain (M-0,0,0, 2, 0).

Histopatholological findings (non- neoplastic):
- Treatment related hyperplasia of the gall bladder mucosa (M-0, 0,0, 1, 2; F-0,0,0,0, 1) and hepatocytomegaly, an enlargement of the cells of the liver (M-0, 0,0,4*, 4*; F-0,0,0, 4*, 4*). The gall bladder hyperplasia, which correlated with gross findings, consisted of increased mucosal volume with taller folds in the mucosa and some arborization of the mucosa.
- Hepatocytomegaly was observed in the 750 ppm group, both sexes, and somewhat less discernible in the 200 ppm dogs. Hepatocytes were larger in diameter than controls and in some cases contained cytoplasm with a granular texture, particularly at the periphery of the morphologic lobule. This appearance, when it occurred, was characteristic of cells containing increased amounts of smooth endoplasmic reticulum. Other cells, occupying the majority of the centrilobular area, contained prominently vacuolated cytoplasm with increased frequency of dark eosinophilic cytoplasmic densities compared to controls. There was a tendency toward more degenerative changes in general in the higher two dose groups.
-The adrenals showed increased frequency and/or severity of vacuolation in the inner zona fasciculata and in the zona reticularis in the 750 ppm group, both sexes. The frequency numbers were not significantly increased over control (M-0, 1, 1, 0, 3; F-3, 1,2, 1,2); however, there was an increase in severity (M-0, 1.0, 1.0,0, 2.7; F-1.0, 1.0, 1.0, 1.0, 2.0).
- Lymphocytic thyroiditis was noted more commonly in the high dose groups (M-0,0, 0, 1,0; F-0, 0, 0, 1, 3), but the change was not statistically significant.
- Other changes noted without relation to treatment included: lymphocytic infiltrate in the gall bladder, renal microliths, various inflammatory conditions in the lungs, lymphoid hyperplasia in the retropharyngeal lymph nodes, parathyroid cysts, mineralization of the meninges covering the spinal cord, fibrosiderotic plaques in the splenic capsule, involution of the thymus (a normal process), and follicular degeneration and c-cell hyperplasia in the thyroid.
: Key result
Dose descriptor:: NOAEL
Effect level:: 20 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: 0.55 mg/kg bw/day (males) / 0.52 mg/kg bw/day (females)
: Key result
Dose descriptor:: LOAEL
Effect level:: 40 ppm
Based on:: test mat.
Sex:: male/female
Basis for effect level:: clinical biochemistry
Remarks on result:: other: corresponding to 1.12 mg/kg bw/day (males) / 1.14 mg/kg bw/day (females)
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 40 ppm
System:: hepatobiliary
Organ:: 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
: 0.52 mg/kg bw/day
Study duration:: chronic
Species:: dog
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:: hepatobiliary
Organ:: liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:: no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:: no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:: short-term repeated dose toxicity: dermal
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 07 February - 21 March 1996
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Reason / purpose for cross-reference:: other: Dose range finding study for short-term study
Qualifier:: according to guideline
Guideline:: OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:: Adopted: 12 May 1991
Deviations:: no
GLP compliance:: yes
Limit test:: no
Species:: rat
Strain:: Wistar
Remarks:: HsdCpb:WU
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Harlan Winkelmann GmbH
- Females: nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks (males), 15 weeks (females)
- Weight at study initiation: 243 to 279 g (males), 217 to 239 g (females)
- Housing: 5 animals (<180g body weight) or 3 animals (>180g body weight) separated by sex in polycarbonate cages type III during adaption period, individally in polycarbonate cages type IIA. Low-dust wood shavings type S 8/15 (Ssniff, Spezialdiaten GmbH, Soest/Westphalia, Germany) were used as litter.
- Diet: Altromin® 1321 meal (Altromin GmbH, Lage), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least seven days

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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 55% ± 5%
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:: semiocclusive
Vehicle:: unchanged (no vehicle)
Remarks:: moistened with water
Details on exposure:: TEST SITE
- Area of exposure: 5.5 x 5.5 cm = 30.25 cm²
- % coverage: >10% body surface area
- Type of wrap if used: gauze-layer ,,Hansapor steril" patch moistened with water, gauze strip was secured by using ,,Peha-Haft" cohesive stretch tape (8 x 23 cm). Additionally, the mobility of the rats was impaired by a ,,Lomir Biomedical Inc." rat jacket, which was connected in two rats with a safety pin to the stretch tape.
- Time intervals for shavings or clipplings: twice weekly

REMOVAL OF TEST SUBSTANCE
- Washing: cleaned with soap and water
- Time after start of exposure: 6h after exposure

TEST MATERIAL
- Amount(s) applied: 50, 250 and 500 mg/kg bw/day
- Constant volume or concentration used: no
- For solids, paste formed: no, moistened with tap water

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes
Analytical verification of doses or concentrations:: no
Details on analytical verification of doses or concentrations:: Analytical determinations (stability, homogeneity) were not performed, because the test substance was applied undiluted and only moistened with water immediately before application.
Duration of treatment / exposure:: 4 weeks exposure + 2 weeks recovery
Frequency of treatment:: 5 days per week for the first three weeks, 6h/day
7 days per week in the fourth week, 6h/day
Dose / conc.:: 50 mg/kg bw/day (actual dose received)
Dose / conc.:: 250 mg/kg bw/day (actual dose received)
Dose / conc.:: 1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:: 5 (main study)
5 (recovery period: control and 1000 mg/kg bw/day)
Control animals:: yes, concurrent no treatment
Details on study design:: - Dose selection rationale: 12 day dose-range finding study (1996d) in female rats. In this study, the doses were 0-100-300-1000 mg/kg. On the application area neither erythema nor oedema of the skin were observed. The administration of 100 and 300 mg/kg showed no toxicologically significant effects. At 1000 mg/kg body weight increased adrenal weights were observed. At all dose levels cholinesterase activity was decreased in erythocytes, plasma and brain.
- Rationale for selecting satellite groups: reversibility of adverse effects
- Post-exposure recovery period in satellite groups: 2 weeks
Observations and examinations performed and frequency:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day, once daily at weekends and public holidays
- Cage side observations checked: body surfaces and orifices, posture, general behavior, breathing and excretory products, including irritation at the dose site, any clinical signs (findings) and abnormalities were recorded

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least twice a day, once daily at weekends and public holidays

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: before start of the study and during the study each day before treatment

BODY WEIGHT: Yes
- Time schedule for examinations: before the study was initiated and at the beginning of each study week

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: 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: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 28 (main groups) and Day 43 (recovery groups)
- Anaesthetic used for blood collection: Yes (diethyl ether)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: leucocytes (leuco), erythrocytes (ery), hemoglobin (HB), hematocrit (HCT), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), platelet count (thro), hepato-quick (hquick) differential blood count: lymphocytes (lym), polymorphonuclear granulocytes (segm), eosinophils (eos), monocytes (mono), normal red blood cells (norm RBC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: glucose test - Day 22/23 (main groups) and Day 41 (recovery groups), Day 28 (main groups) and Day 43 (recovery groups)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: aspartat aminotransferase (ASAT), alkaline phosphatase (APh), alanine aminotransferase (ALAT), , glutamate dehydrogenase activities (GLDH), gamma-glutamyl transpeptidase activity (γGT), cholesterol (Chol), triglycerides (trigl), creatinine (crea), urea, bilirubin total (bible-t), protein total (prot), albumin, sodium (Na), potassium (K), calcium (Ca), chloride (Cl), phosphorus (P), glucose, cholinesterase activity in plasma (CHE), cholinesterase activity in erythrocytes (CHE/E) and cholinesterase activity in brain (CHE/B)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: No
Sacrifice and pathology:: GROSS PATHOLOGY: Yes
- Organ weights: adrenal glands, brain, heart, kidneys, liver, lung, spleen, testes, thymus

HISTOPATHOLOGY: Yes (adrenal glands, brain, heart, kidneys, liver, skin (treated and untreated), spleen, testes, thymus, thyroid gland with parathyroid gland, urinary bladder, physical identifier (tattooed ears), one liver lobe and lungs and all organs or tissues with macroscopic findings)
Statistics:: The quantitative results for individual animals were used to calculate arithmetic group means and standard deviations. The results for the groups that received the test substance were compared with those for the control group and significant differences indicated by "+" for p<0.05 and "++" for p<0.01.
The Dunnett test:body weight, feed consumption and organ weight data (relative organ weights subsequent to logarithmic transformation).
The clinical findings are presented by individual animal findings with information on the time of occurrence in question.
Calculation of means and variances was based in part on the non-rounded original values.
Clinical signs:: no effects observed
Description (incidence and severity):: no effects observed
Dermal irritation:: no effects observed
Description (incidence and severity):: no effects observed
Mortality:: no mortality observed
Description (incidence):: no mortality observed
Body weight and weight changes:: no effects observed
Description (incidence and severity):: For details please refer to Tables 1 and 2 in "any other information on results incl. tables".
Food consumption and compound intake (if feeding study):: no effects observed
Description (incidence and severity):: For details please refer to Tables 3 and 4 in "any other information on results incl. tables".
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: not examined
Haematological findings:: effects observed, non-treatment-related
Description (incidence and severity):: 250 mg/kg bw/day: decreased MCV in females
1000 mg/kg bw/day: decreased MCV in females; no correlation with the doses administered and with the other sex, and all individual values were within the 2 standard deviation-range of the historical control values

For details please refer to Tables 5 in "any other information on results incl. tables".
Clinical biochemistry findings:: effects observed, treatment-related
Description (incidence and severity):: 1000 mg/kg bw/day: decreased ChE activity in erythrocytes in both sexes, in brain in females only (reversible), increased protein and albumin levels in males (reversible)

For details please refer to Tables 6 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, non-treatment-related
Description (incidence and severity):: 1000 mg/kg bw/day: increased thymus and testes weights at end of treatment period, but values were comparable to the controls of the recovery group (in the recovery group all organ weights were comparable to controls)

For details please refer to Tables 7 in "any other information on results incl. tables".
Gross pathological findings:: no effects observed
Description (incidence and severity):: no effects observed
Neuropathological findings:: not examined
Histopathological findings: non-neoplastic:: no effects observed
Description (incidence and severity):: no effects observed
Histopathological findings: neoplastic:: no effects observed
Description (incidence and severity):: no effects observed
Other effects:: not examined
: Key result
Dose descriptor:: NOAEL
Remarks:: local
Effect level:: 1 000 mg/kg bw/day (actual dose received)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: no adverse effects observed up to this dose level
: Key result
Dose descriptor:: NOAEL
Remarks:: systemic
Effect level:: 250 mg/kg bw/day (actual dose received)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: clinical biochemistry
: Key result
Dose descriptor:: LOAEL
Remarks:: systemic
Effect level:: 1 000 mg/kg bw/day (actual dose received)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: clinical biochemistry
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 1 000 mg/kg bw/day
System:: nervous system
Organ:: other: ChE activity inhibited in erythrocytes and brain (statistically significant inhibition by 20% or more in erythrocytes or brain is considered a clear toxicological effect)
Treatment related:: yes
Dose response relationship:: not specified
Relevant for humans:: not specified

Endpoint conclusion

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 250 mg/kg bw/day
Study duration:: subacute
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:: nervous system
Organ:: blood; other: Cholinesterase activity

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:: short-term repeated dose toxicity: dermal
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 07 February - 21 March 1996
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Reason / purpose for cross-reference:: other: Dose range finding study for short-term study
Qualifier:: according to guideline
Guideline:: OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:: Adopted: 12 May 1991
Deviations:: no
GLP compliance:: yes
Limit test:: no
Species:: rat
Strain:: Wistar
Remarks:: HsdCpb:WU
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Harlan Winkelmann GmbH
- Females: nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks (males), 15 weeks (females)
- Weight at study initiation: 243 to 279 g (males), 217 to 239 g (females)
- Housing: 5 animals (<180g body weight) or 3 animals (>180g body weight) separated by sex in polycarbonate cages type III during adaption period, individally in polycarbonate cages type IIA. Low-dust wood shavings type S 8/15 (Ssniff, Spezialdiaten GmbH, Soest/Westphalia, Germany) were used as litter.
- Diet: Altromin® 1321 meal (Altromin GmbH, Lage), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least seven days

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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 55% ± 5%
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:: semiocclusive
Vehicle:: unchanged (no vehicle)
Remarks:: moistened with water
Details on exposure:: TEST SITE
- Area of exposure: 5.5 x 5.5 cm = 30.25 cm²
- % coverage: >10% body surface area
- Type of wrap if used: gauze-layer ,,Hansapor steril" patch moistened with water, gauze strip was secured by using ,,Peha-Haft" cohesive stretch tape (8 x 23 cm). Additionally, the mobility of the rats was impaired by a ,,Lomir Biomedical Inc." rat jacket, which was connected in two rats with a safety pin to the stretch tape.
- Time intervals for shavings or clipplings: twice weekly

REMOVAL OF TEST SUBSTANCE
- Washing: cleaned with soap and water
- Time after start of exposure: 6h after exposure

TEST MATERIAL
- Amount(s) applied: 50, 250 and 500 mg/kg bw/day
- Constant volume or concentration used: no
- For solids, paste formed: no, moistened with tap water

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes
Analytical verification of doses or concentrations:: no
Details on analytical verification of doses or concentrations:: Analytical determinations (stability, homogeneity) were not performed, because the test substance was applied undiluted and only moistened with water immediately before application.
Duration of treatment / exposure:: 4 weeks exposure + 2 weeks recovery
Frequency of treatment:: 5 days per week for the first three weeks, 6h/day
7 days per week in the fourth week, 6h/day
Dose / conc.:: 50 mg/kg bw/day (actual dose received)
Dose / conc.:: 250 mg/kg bw/day (actual dose received)
Dose / conc.:: 1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:: 5 (main study)
5 (recovery period: control and 1000 mg/kg bw/day)
Control animals:: yes, concurrent no treatment
Details on study design:: - Dose selection rationale: 12 day dose-range finding study (1996d) in female rats. In this study, the doses were 0-100-300-1000 mg/kg. On the application area neither erythema nor oedema of the skin were observed. The administration of 100 and 300 mg/kg showed no toxicologically significant effects. At 1000 mg/kg body weight increased adrenal weights were observed. At all dose levels cholinesterase activity was decreased in erythocytes, plasma and brain.
- Rationale for selecting satellite groups: reversibility of adverse effects
- Post-exposure recovery period in satellite groups: 2 weeks
Observations and examinations performed and frequency:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day, once daily at weekends and public holidays
- Cage side observations checked: body surfaces and orifices, posture, general behavior, breathing and excretory products, including irritation at the dose site, any clinical signs (findings) and abnormalities were recorded

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least twice a day, once daily at weekends and public holidays

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: before start of the study and during the study each day before treatment

BODY WEIGHT: Yes
- Time schedule for examinations: before the study was initiated and at the beginning of each study week

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: 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: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 28 (main groups) and Day 43 (recovery groups)
- Anaesthetic used for blood collection: Yes (diethyl ether)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: leucocytes (leuco), erythrocytes (ery), hemoglobin (HB), hematocrit (HCT), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), platelet count (thro), hepato-quick (hquick) differential blood count: lymphocytes (lym), polymorphonuclear granulocytes (segm), eosinophils (eos), monocytes (mono), normal red blood cells (norm RBC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: glucose test - Day 22/23 (main groups) and Day 41 (recovery groups), Day 28 (main groups) and Day 43 (recovery groups)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: aspartat aminotransferase (ASAT), alkaline phosphatase (APh), alanine aminotransferase (ALAT), , glutamate dehydrogenase activities (GLDH), gamma-glutamyl transpeptidase activity (γGT), cholesterol (Chol), triglycerides (trigl), creatinine (crea), urea, bilirubin total (bible-t), protein total (prot), albumin, sodium (Na), potassium (K), calcium (Ca), chloride (Cl), phosphorus (P), glucose, cholinesterase activity in plasma (CHE), cholinesterase activity in erythrocytes (CHE/E) and cholinesterase activity in brain (CHE/B)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: No
Sacrifice and pathology:: GROSS PATHOLOGY: Yes
- Organ weights: adrenal glands, brain, heart, kidneys, liver, lung, spleen, testes, thymus

HISTOPATHOLOGY: Yes (adrenal glands, brain, heart, kidneys, liver, skin (treated and untreated), spleen, testes, thymus, thyroid gland with parathyroid gland, urinary bladder, physical identifier (tattooed ears), one liver lobe and lungs and all organs or tissues with macroscopic findings)
Statistics:: The quantitative results for individual animals were used to calculate arithmetic group means and standard deviations. The results for the groups that received the test substance were compared with those for the control group and significant differences indicated by "+" for p<0.05 and "++" for p<0.01.
The Dunnett test:body weight, feed consumption and organ weight data (relative organ weights subsequent to logarithmic transformation).
The clinical findings are presented by individual animal findings with information on the time of occurrence in question.
Calculation of means and variances was based in part on the non-rounded original values.
Clinical signs:: no effects observed
Description (incidence and severity):: no effects observed
Dermal irritation:: no effects observed
Description (incidence and severity):: no effects observed
Mortality:: no mortality observed
Description (incidence):: no mortality observed
Body weight and weight changes:: no effects observed
Description (incidence and severity):: For details please refer to Tables 1 and 2 in "any other information on results incl. tables".
Food consumption and compound intake (if feeding study):: no effects observed
Description (incidence and severity):: For details please refer to Tables 3 and 4 in "any other information on results incl. tables".
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: not examined
Haematological findings:: effects observed, non-treatment-related
Description (incidence and severity):: 250 mg/kg bw/day: decreased MCV in females
1000 mg/kg bw/day: decreased MCV in females; no correlation with the doses administered and with the other sex, and all individual values were within the 2 standard deviation-range of the historical control values

For details please refer to Tables 5 in "any other information on results incl. tables".
Clinical biochemistry findings:: effects observed, treatment-related
Description (incidence and severity):: 1000 mg/kg bw/day: decreased ChE activity in erythrocytes in both sexes, in brain in females only (reversible), increased protein and albumin levels in males (reversible)

For details please refer to Tables 6 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, non-treatment-related
Description (incidence and severity):: 1000 mg/kg bw/day: increased thymus and testes weights at end of treatment period, but values were comparable to the controls of the recovery group (in the recovery group all organ weights were comparable to controls)

For details please refer to Tables 7 in "any other information on results incl. tables".
Gross pathological findings:: no effects observed
Description (incidence and severity):: no effects observed
Neuropathological findings:: not examined
Histopathological findings: non-neoplastic:: no effects observed
Description (incidence and severity):: no effects observed
Histopathological findings: neoplastic:: no effects observed
Description (incidence and severity):: no effects observed
Other effects:: not examined
: Key result
Dose descriptor:: NOAEL
Remarks:: local
Effect level:: 1 000 mg/kg bw/day (actual dose received)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: no adverse effects observed up to this dose level
: Key result
Dose descriptor:: NOAEL
Remarks:: systemic
Effect level:: 250 mg/kg bw/day (actual dose received)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: clinical biochemistry
: Key result
Dose descriptor:: LOAEL
Remarks:: systemic
Effect level:: 1 000 mg/kg bw/day (actual dose received)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: clinical biochemistry
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 1 000 mg/kg bw/day
System:: nervous system
Organ:: other: ChE activity inhibited in erythrocytes and brain (statistically significant inhibition by 20% or more in erythrocytes or brain is considered a clear toxicological effect)
Treatment related:: yes
Dose response relationship:: not specified
Relevant for humans:: not specified

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 7.5 mg/cm²
Study duration:: subacute
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.

Additional information

Repeated dose toxicity: oral

Mouse

- chronic study, 2 years

In this carcinogenicity study in B6C3F1 mice conducted according to OECD 451, the test substance was administered in rodent diet at concentrations of 20, 100, 500, and 2000 ppm to groups of 50 male and female (1997n) for 24 months. Additional 20 animals were administered diet containing 0 or 2000 ppm test substance and 10 animals of each group were used for interim investigations in Weeks 27 and 53 to study gallbladder lesions in time. The mean test substance intake was 5.4, 28.0, 130.6 and 575.4 mg/kg bw/day for males and 7.7, 41.9, 201.2 and 831.1 mg/kg bw/day for females.

No test substance related clinical findings were observed and mortalities in treatment and control groups were comparable. At 2000 ppm lower body weights (9.6% in Week 79 in main groups or 13.6% in Week 45 in satellite groups) were observed in males, whereas body weights of females were only slightly reduced (at a maximum of 7.1% in Week 47). Slightly more food was consumed in all treatment groups compared to controls. Haematological analysis did not reveal treatment-related effects on red and white blood cell counts and effects were observed on haematopoietic organs/tissues up to 2000 ppm. Determination of cholinesterase (ChE) activity revealed a statistically significant inhibition of cholinesterase in plasma of females at 2000 ppm. Further, a statistically significant inhibition of cholinesterase by more than 20% was determined in females in week 54 in erythrocytes, but not at termination. In males inhibiton of cholinesterase by more than 20% was also observed at 500 and 2000 ppm in week 54, not reaching statistically significance.The inhibiton of cholinesterase in plasma is although being significantnot considered biologically relevant according to ECHA guidance Chapter R7a (2016). Although statistically significant inhibition by 20% or more in erythrocytes or brain is considered a toxicologically relevant effect, thebiological relevance of the observed cholinesterase inhibiton in erythrocytes is questionable, since it was observed at only one time point. Thus, based on the available data, the observed effects on ChE activity in mice is not interpreted as indicative of a clear toxicological effect.

At termination slightly reduced plasma glucose values were observed in females at 500 or 2000 ppm. Since there was no correlate to this finding, these very slight deviations from controls were not considered toxicologically relevant. At a dose level of 2000 ppm, elevated absolute and relative kidney weights were observed in females in Week 54 and elevated absolute weights in males at terminal necropsy. Since any biochemical or morphological correlates were missing in females the findings were considered incidental. Differences in males were very small and could be explained by slight differences in body weights. In the 2000 ppm group relative higher testes weights compared to control were observed in Weeks 28, 54 and 106-108. Since morphological changes were absent and the deviations from control values were small (<8%), no toxicological relevance was attributed to this finding. Statistically significant deviations in absolute organ weights of spleen in males at terminal necropsy were very small and could be explained by slight differences in body weights. No remarkable differences were evident in brain weights in Weeks 28, 54 and at terminal necropsy. Absolute (females) and relative liver weights (both sexes) were significantly elevated at interim evaluation in the highest dose group (2000 ppm). Also at terminal necropsy relative liver weights of females were increased at a dose of 500 and 2000 ppm. Clinical biochemistry analysis showed slight effects on the liver function such as elevated plasma levels of cholesterol (females) and reduced triglyceride values (males) beginning at 500 ppm. In line with these findings, histopathology revealed adverse effects on the liver at 2000 ppm. In Week 28, hepatocytes of males and females showed cytoplasmic changes (eosinophilic and larger cells with larger nuclei) in males and females. In addition, one female exhibited a focal necrosis and one male showed a basophilic focus in the liver. In Week 54 nearly all mice showed hepatocellular cytoplasmic changes in the highest dose group. At terminal necropsy increased liver weights were also visible in 500 and 2000 ppm females. However, there were no treatment-related changes in the liver morphology of mice scheduled for a 24-month treatment. Taken together these findings indicated a changed liver function as a result of an adaptive response of the liver beginning at 500 ppm.

At a dose of 2000 ppm gross pathology revealed adverse effects on the gallbladder during interim evaluations in Weeks 28 and 54 and at 500 and 2000 ppm at terminal necropsy. In line with these gross pathology findings a simple or cystic mucosa hyperplasia of the gallbladder epithelium was observed in 500 ppm females and 2000 ppm males and females. Additionally performed investigations (1997i) indicated that an increase in bile acid synthesis is responsible for these morphological alterations and for changes in some clinical pathological parameters such as plasma cholesterol and triglyceride levels.

Clinical laboratory, gross and histopathological investigations as well as organ weight analysis did not indicate toxicity to other organs in the investigated dose range.

The number of mice with benign or malignant neoplasms of the nervous system, urinary tract, male reproductive system, gastrointestinal tract, bone marrow, spinal cord, and muscular-skeletal system was zero or low in all groups. In the groups 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 the control and 20 ppm groups. 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 was not assumed for this tumour type. Taking into account data from a two year study with B6C3F1 mice which was 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) carcinogenicity 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 was only slightly above the reference range of studies performed 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. No remarkable differences were visible in remaining tumour incidences between treated and untreated mice. There was no toxicologically relevant shift in the tumour spectrum or time of tumour occurrence. No increase in the total number of tumours or the incidence of animals with tumours was visible up to 2000 ppm. Therefore the test substance was considered to be not carcinogenic in mice. In conclusion there was no treatment related shift in the tumour range and no increase in the total number of tumours or animals with tumours which can be attributed to a carcinogenic potential of the test substance.

Based on these findings a non-neoplastic NOAEL of 100 ppm (corresponding to 28.0 and 41.9 mg/kg bw/day) and a neoplastic NOAEL of 2000 ppm (corresponding to 575.4 and 831.1 mg/kg bw/day) was determined for male and female mice, respectively.

- evaluation of gall bladder effects in mice

An additional expert evaluation of gall bladder effects, which were observed in a carcinogenicity study in mice (1997n) was performed (2000d).

Liver and gallbladder sections from 50 male and 50 female mice treated at a high-dose level of 2000 ppm for 24 months were stained with haematoxylin and eosin for specific evaluation of gallbladder effects. In addition liver and gallbladder sections from interim sacrifice groups of the above study treated over a 28-week and a 57-week period were investigated.

Many of the animals dosed at 2000 ppm over a period of 24-month showed hyperplasia of the gall bladder, which was observed more frequent in females than in males. The hyperplasia was characterized by a thickening of the mucosa and formation of areas with cystic structures with a severity ranking from mild to marked. There was no evidence of tumour growth in the gallbladder in any investigated animal.

For classification of gallbladder lesions the following classification, defining epithelial tumours of the mouse gallbladder was consulted for the additional evaluation (Draft version International Classification of Rodent Tumours. Part II: The Mouse. Springer-Verlag, Heidelberg, in press):

1. Adenoma (synonym: papillary adenoma)

• Growth is disordered, predominantly papillary or cauliflower-like.

• Amount of fibrovascular stroma is variable.

• Epithelium is single-layered, but may also be multi-layered.

• Cells may exhibit moderate atypia, with enlarged nuclei or even giant nuclei with one or two nucleoli.

• Mitotic figures are present.

• Often inflammatory cellular infiltration and focal mineralization of the stroma may be present.

2. Adenocarcinoma

• May be a sessile broad-based mass or a diffuse thickening of the mucosa.

• Invasion of the wall of the gallbladder or adjacent tissue may occur.

• Growth pattern is disordered.

• Cellular atypia is present. Cytoplasm is scant and basophilic.

• Mitotic figures are present.

Tumours of the gall bladder should be differentiated from adenomatoid change, which is noted in aging mice of different strains. Adenomatoid change consists of a thickened mucosa with gland formation. The hypertrophic cells contain eosinophilic crystalline inclusions which may also be found in the lumen of the glands and hi the gallbladder. The cells are well differentiated with no or only little atypia.

The observed gallbladder lesions consisted of a hyperplastic mucosa resembling adenomatoid change. There was no papillary structure with a fibrovascular stalk as observed in papillary adenomas and no infiltrative growth with cellular atypia. Mitoses and/or basophilia of the cytoplasm which would give evidence of malignancy were not present. Also at the interim evaluation in animals treated for 28 or 57 weeks, hyperplasia of the gall bladder was present, but without any indication of neoplastic growth. Staining of mucinous substances with Alcian blue of sections from the interim evaluation in Week 28 and 47, revealed mucinous substances in the gallbladder of treated (2000 ppm) animals, especially in females.

It was concluded that administration of the test substance in a chronic/oncogenicity study produced hyperplasia in the gall bladder mucosa of mice. In the additional evaluation, no gall bladder tumours were found and therefor no neoplastic growth was evident.

-sub-chronic study, 90 days

A sub-chronic 15 week range-finding study for a two year study in B6C3F1 mice was performed GLP conform and according to OECD 408 (1997h). Ten animals per sex and treatment level received a diet with the test substance concentrations of 20, 100, 600, 3600 and 7200 ppm. The average daily test substance intake was 8.1, 42.4, 266.3, 1593.1 and 3386.2 mg/kg bw/day in males and 10.6, 55.2, 331.8, 1877.1 and 3943.3 mg/kg bw/day in females.

Mortalities were not observed up to 7200 ppm and no particular clinical findings occurred in treated mice. Males receiving doses of test substance of 600 ppm and above had reduced bodyweights, whereas food consumption was increased at 7200 ppm. Water intake was not influence by the treatment. Haematology analysis did not reveal toxicologically relevant findings on red or white blood cell parameters. Observed differences to controls were considered not treatment-related and non-adverse, since they were not concentration-level related and/or very small.

Clinical chemistry analysis revealed inhibition of cholinesterase activity (ChE) in the plasma at levels of 3600 ppm and above. Observations at 100 and 600 ppm were not considered of toxicological relevance as the inhibition was only < 13%. In erythrocytes no inhibition of ChE was observed. Other clinical chemistry parameters such as activities of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase, plasma or blood concentrations of glucose, total protein, albumin, total bilirubin, urea and creatinine were not altered in a toxicologically relevant manner. Observed significant deviations were considered to be not treatment-related, since differences from controls were very small and dose independently distributed.

At dose levels of 3600 and above signs of changed liver function included increased liver weights, higher cholesterol and decreased triglyceride levels in plasma and morphological alterations such as hypertrophic hepatocytes and single cell necrosis, indicating that the liver is the target organ.

Gross pathology and histopathology analysis revealed black discolouration of gall bladders (3600 and 7200 ppm) and hypersecretory and hyperplastic changes at doses of 600 ppm and above. In most of these mice the lumen was filled with an eosinophilic concrement. Additionally, crystal formation could be detected in one 3600 ppm male and four 7200 ppm mice. In addition, in mice dosed at 3600 and 7200 ppm focal inflammatory infiltrations were seen in subepithelial cell layers as well. The abnormal and probably irritating bladder content was assumed to be responsible for morphological alterations in the gallbladder.

Further findings were reduced cytoplasmatic vacuoles in the proximal tubules of the kidneys in male mice dosed at 600 ppm and above and a reduction of vacuoles in cells of the x-zone of the adrenals in females dosed at 7200 ppm. It was assumed that the absence of vacuoles in the proximal tubules of the kidneys is a secondary finding possibly resulting of an altered liver function and a direct adverse effect on the kidneys was not considered. The toxicological relevance of vacuoles in adrenal cells was considered questionable. Gross pathology, organ weight determination and histopathology analysis did not evidence adverse effects in further organs of mice treated up to 7200 ppm.

Based on these findings gallbladder and liver were considered primary target organs for the test substance and a NOAEL of 100 ppm (corresponding to 42.4 and 55.2 mg/kg bw/day in males and females, respectively) was determined.

- sub-acute study, 4 weeks

A sub-acute 4 week study with a 2 week recovery period was performed in B6C3F1 mice similar to OECD guideline 407 (1998c). The test substance solved in olive oil was administered per diet. Six animals per sex and dose received dietary concentrations of 100, 1000 and 10000 ppm. The average daily test substance intake was 19.9, 224.4 and 3421 mg/kg bw/day in males and 22.5, 253.7 and 2984 mg/kg bw/day in females. The additional recovery groups consisted of control animals and animals dosed at 10000 ppm. Controls received diet with 0.8% olive oil only. Examinations included clinical signs, determination of bod weight, food and test substance intake, food efficiency, haematological and clinical biochemistry analysis. At necropsy gross pathology examinations, determination of organ weights and histopathology analysis were performed.

Mortalities or clinical findings were not observed up to the highest dietary concentration of 10000 ppm.

At 10000 ppm body weight and food consumption and efficiency were significantly decreased in males and females.

Haematology analysis revealed a significant decrease of haematocrit and haemoglobin level, erythrocyte counts and mean corpuscular cell volume (MCV) at 1000 and 10000 ppm in females. The observed decreased platelet count at 100 and 10000 ppm in males was not considered treatment-related, since dose dependency was missing. No histopathological findings were observed in haematopoietic organs.

In chemical biochemistry analysis total cholesterol levels were significantly and increased in males at all dose levels and females at 1000 and 10000 ppm in a dose dependent manner. Whereas triglyceride levels were decreased at dose levels of 100 ppm and above in females, only. These findings suggest a test substance-related effect on lipid metabolism in mice. Furthermore, cholinesterase activity was significantly decreased (> 20%) in blood of females of the highest dose group. However, since no distinction was made between plasma and erythrocytes, no conclusive decision on the biological relevance of this effect can be drawn. Other changes in clinical biochemistry parameters increase of blood urea nitrogen (BUN) in males, and increase in total protein in both sexes and reduced albumin levels in males and increased albumin levels in females, did not show dose-dependency and were therefore considered non-treatment-related.

Kidney weights (absolute and relative) were significantly decreased in males also during the recovery period. But there were no further indications for toxicity detectable in clinical biochemistry or histopathology analysis.

At the highest dose level significantly decreased absolute and relative ovary weights were observed, but histopathology findings.

Gross pathology and histopathology analysis clearly indicated toxic effects on the liver. Dark colouration was observed at the highest dose level in males and females and absolute and relative liver weights were significantly increased in females at 1000 ppm and above and in males of the highest dose group. In line with these findings irregular distribution of eosinophilic body in hepatocytes was found at all dose levels in females and at 10000 ppm in all males. In addition mitosis of hepatocytes was found in one female. However, clinical biochemistry data did not clearly indicate changes in liver function. Alkaline phosphatase levels were increased in males at all dose levels, but decreased in females of the highest dose group. Further parameters such as glutamat-oxalacetat-transaminase, glutamat-pyruvat-transaminase were not changed in both sexes. Analysis of the recovery showed that the adverse liver effect were partially reversible. In males and females, eosinophilic body in hepatocytes disappeared at the end of the recovery period. Also the incidence of dark colouration of the liver was remarkably reduced. However, relative liver weights were still increased in males and focal necrosis in liver was observed in 2/6 treated mice and swelling of hepatocytes and fatty droplets in hepatocytes was observed in1/6.

Histopathology also revealed proliferation of the epithelial mucosa in gallbladder in males of all dose groups and in females at 1000 ppm and above. Effects on gallbladder were still observed in one male of the recovery group. A further finding was vacuoles of thyroid glands in two males of the highest dose group.

Since adverse effects especially on liver and gallbladder were observed at all dose levels a NOAEL could not be determined in this study.

- sub-acute study, 14 days

A sub-acute 2 week study was performed in male and female ICR mice (1993a). The test substance solved in 1% Cremophor EL DW was administered per gavage. Six control animals and six or seven animals per sex and dose received 1000 and 25000 mg/kg bw/day. Examinations included clinical signs, mortalities, determination of bod weight, haematological and clinical biochemistry analysis and an autopsy.

Mortalities, clinical findings or effects on body weight were not observed up to the highest dose tested. At a dose of 25000 mg/kg bw/day white blood cells were significantly increased in females. Other haematological findings were not observed. Clinical biochemistry revealed a significant increase in total protein in males receiving 1000 mg/kg bw/day only. At autopsy of the animals on Day 15 a statistically significant increase of liver weights was observed in males receiving 1000 and 2500 mg/kg bw/day and in females receiving 1000 mg/kg bw/day. In addition, in the highest dose group spleen weight in females and thymus weights in males were significantly increased. Based on these findings a NOAEL was not determinable.

Dog

- chronic study, 1 year

A chronic one-year study in beagle dogs was performed according to OECD 452. Four animals per sex and treatment level received a diet with the nominal test substance concentrations of 20, 40, 200 and 750 ppm (1997e). The average daily test substance intake was 0.55, 1.12, 5.35 and 24.32 mg/kg bw/day in males and 0.52, 1.14, 5.50 and 24.74 mg/kg bw/day in females.

Examinations included clinical observations, determination of body weight, food consumption, food efficiency, test substance intake, opthalmoscopic examinations, haematology and clinical chemistry analysis, urinalysis, gross and histopathological examinations in different organs and tissues. Additionally to guideline requirements, cardiac and neurologic effects were investigated and included electrocardiography, blood pressure measurements, peripheral and cranial reflex tests, task performance tests, gait and behavioural observations, as well as rectal temperature measurements.

No treatment-related clinical findings, mortalities, opthalmoscopic findings or effects on urinalyses were observed at any dose level. During course of the study a decrease in body weights and body weight gain was observed in males and females at 750 ppm. At the same dose a trend of increased food uptake was noticed compared to corresponding controls.

Haematology analysis revealed an increase in platelet counts at 750 ppm in both sexes. Although an increase was also observed at other dose levels in males, only the change at 750 ppm is believed to be treatment-related, as all other groups followed the control pattern of a gradual decrease over time, while the 750 ppm group increased over time.

The Phase I-inducible hepatic enzymes, N-demethylase and O-demethylase were significantly increased at all dose levels in both sexes. At the 20 ppm level N- and O-demethylase were increased, but within or at the upper limit of the historical control range. Since histopathological changes were not observed at 20 or 40 ppm, the degree of Phase I-induction at this dose levels was not considered as adverse effect, but an expected physiological response to detoxification. Clinical biochemistry also revealed a cytochrome P-450 increase in the 200 and 750 ppm treatment groups in both sexes and at 40 ppm in females. Further liver enzyme changes included: an increase of alanine aminotransferase at 750 ppm in both sexes and at 200 ppm in females at several intervals, an increase of alkaline phosphatase at 200 and 750 ppm in both sexes and in females of the 40 ppm group at six months and later, and an increase of gamma-glutamyl transpeptidase at 750 ppm group in both sexes at most intervals and at the six-month interval in females of the 200 ppm dose group.

Albumin and total protein were decreased in the 750 ppm dose group in both sexes at most intervals and were attributed to liver functionality. Other clinical chemistry changes included a decrease of calcium in 750 ppm females at 3, 9, and 12 months and in 200 ppm females at three months, an increase of triiodothyronine and thyroxine in males of the 750 ppm group at 12 months only. The central role of the liver in clearance and detoxification of test substance was considered to influence clearance of thyroid hormone and thereby altering the circulating levels of T3 and T4 through increased sulfatation and glucuronidation, respectively [1].

Gross necropsy examinations revealed thickening of the gallbladder at 750 ppm in males and enlargement of the liver in one male and one female of the 750 ppm dose group. At the same dose absolute and relative liver weights in males and relative liver weights in females were significantly increased. Also in males of the 40 ppm dose group an increase in relative liver weights was observed.

In addition, a non-significant increase in hepatic weights was detected in females of the 40 ppm group and in both sexes of the 200 ppm group. Absolute thyroid weights were observed in females of the 200 and 750 ppm dose group. Relative thyroid weights were significantly increased at 750 ppm in both sexes and at 20 and 200 ppm in females, only. Furthermore, adrenals were slightly heavier at 750 ppm in both sexes, however statistical significance was only observed in males. Other gross pathology observations, pituitary cysts, raised/discoloured zones on the spleen, reduced thymus size and dilated ventricles in the brain were not considered test substance related.

Histopathology findings included hepatocytomegaly and hyperplasia of the gallbladder at 200 and 750 ppm in both sexes, an increased frequency and/or severity of vacuolation in the inner zona fasciculata and in the zona reticularis of adrenals at 750 ppm in both sexes and an non-significant increase of thyroiditis in both sexes at 200 ppm and females also at 750 ppm. Gallbladder findings were considered to be influenced by liver toxicity. The liver and gallbladder are functionally and physiologically coordinated in the synthesis, transport and secretion of bile acids [2]. Gallbladder mucosal changes were considered likely to be a compensatory response to increased bile acid flow.

Other observed effects like lymphocytic infiltrate in the gallbladder, renal microliths, various inflammatory conditions in the lungs, lymphoid hyperplasia in the retropharyngeal lymph nodes, parathyroid cysts, mineralization of the meninges covering the spinal cord, fibrosiderotic plaques in the splenic capsule, involution of the thymus, follicular degeneration and c-cell hyperplasia in the thyroid were not considered treatment-related.

The additional neurologic and cardiac analysis did not reveal any treatment-related adverse neurological effects and no dose-related changes on electrocardiography or blood pressure parameters.

A NOAEL was established at 20 ppm (corresponding to 0.55 and 0.52 mg/kg bw/day in males and females, respectively) and was based on adverse effects in the liver. At the 20 ppm dose level N- and O-demethylase were within or at the upper limit of the historical control range in both sexes. No histopathological changes were seen at 20 ppm, supporting the conclusion that this degree of Phase I-induction was not an adverse effect, rather an expected physiological response to detoxification. The 40 ppm dose level was assumed to be the threshold of toxicity, since alkaline phosphatase and Phase I enzymes were increased.

References:

[1] Capen C. C., 1995 Toxic Responses of the Endocrine System, In: Casarett and Doull's Toxicology: The Basic Science of Poisons, 5th Edition (Ed.: CD Klaassen). MacGraw-Hill, New York, NY pp. 617-640

[2] Hofman A. F., 1988 Bile Acids, In: The Liver - Biology and Pathobiology, (Eds.: IM Arias, WB Jakoby, H Popper, D Schachter and DA Shafritz). Raven Press, New York, NY pp. 553-572

- sub-chronic study, 90 days

A sub-chronic 90 days study in beagle dogs was performed GLP-conform according to OECD 409 (1996c). Four animals per sex and treatment level received a diet with the nominal test substance concentrations of 75, 300 and 1200 ppm. The average daily test substance intake was 2.98, 12.36 and 45.54 mg/kg bw/day in males and 2.95, 12.41 and 43.20 mg/kg bw/day in females.

Examinations included clinical observations, determination of body weight, food consumption, test substance intake, opthalmoscopic examinations, haematology and clinical chemistry analysis, urinalysis and gross and histopathology examinations in different organs and tissues. Additionally to guideline requirements, cardiac and neurologic effects were investigated and included electrocardiography, blood pressure measurements, heart rate, mental status/behaviour, gait characteristics, postural status and reactions, and spinal/cranial reflex tests.

Treatment-related effects on clinical signs, ophthalmology or neurology were not observed and mortalities occurred at any dose level. In the highest dose group food consumption and body weight was statistically significant reduced in males and a similar trend was also detectable in females. Analysis of haematology parameters revealed a relative neutrophil (seg):lymphocyte reversal in 1200 ppm females. Since the absolute lymphocyte count was higher in females at 1200 ppm at the three month interval and in males at the one month interval, the observed reversal of neutrophil (seg):lymphocyte ratio was considered to be based on lymphocytosis.

Electrocardiography analysis revealed a statistical decrease in 1200 ppm males in P-wave amplitude and P-wave duration when measured just prior to study termination. In addition there was a decrease in R-wave amplitude in females dosed at 300 and 1200 ppm compared to controls. Determination of heart weights showed a decrease in 1200 ppm females. However, histopathological changes, arrhythmias, changes in blood pressure parameters or heart rate were not observed. Considering that there was decreased food consumption and body weight in the 1200 ppm group, the electrocardiographic effects were ascribed to poor nutrition and not considered due to a direct myocardial insult.

Clinical chemistry revealed the liver as a target organ, since hepatocellular enzymes were changed. Alanine aminotransferase was increased in the 1200 ppm group in both sexes at all intervals. Alkaline phosphatase was increased in the 300 and 1200 ppm dose groups in both sexes, except in 300 ppm males at one month. In addition gamma-glutamyl transpeptidase was elevated in the 1200 ppm group in males at two months and females at two and three months. Also aspartate aminotransferase levels were slightly increased in 1200 ppm males and females, but there was a wide variation in individual responses.

In addition, functional parameters associated with liver changes were observed and included increased bile acids in the 1200 ppm group in males at two and three months and in females at one month, decreased albumin in the 1200 ppm group in both sexes at all intervals, decreased total protein in the 1200 ppm group in both sexes at all intervals and in 300 ppm females at two and three months, decreased cholesterol in the 1200 ppm group in both sexes at all intervals and decreased triglycerides in the 1200 ppm group in both sexes at the two and three months interval. The increased N-and O-demethylase in all treatment groups in both sexes and the increased cytochrome P-450 in 75 and 300 ppm females revealed the expected induction response to detoxification of the test substance in the liver.

Furthermore triiodothyronine and thyroxine were decreased in the 1200 ppm group in both sexes at the two and three month interval. Based on liver induction and increased biliary dynamics an extrathyroidal mechanism was suggested. A primary thyroidal mechanism was considered unlikely since there were no microscopic changes.

An additional clinical chemistry finding was a calcium decrease in the 1200 ppm group in both sexes at all intervals, in the 300 ppm group in males at one and three month; in 300 ppm dosed females at two and three months and in 75 ppm dosed males at one and three months. Only effects observed at 300 and 1200 ppm were outside the limits of historical control data and considered biologically relevant. The decrease of calcium was considered likely secondary to the decreased serum albumin noted above, as hypocalcemia is known to occur in hypoalbuminemic dogs.

Gross necropsy examination and histopathology revealed thickened gallbladders and hyperplasia of gallbladder mucosa in the 1200 ppm dose group in both sexes. Hyperplasia of the gallbladder mucosa was also observed in females of the 300 ppm group. The gallbladder mucosa changes were discussed as a potentially compensatory response to changes in bile composition, and not as direct pathologic change in that tissue. Since the liver and gallbladder are functionally and physiologically coordinated in the synthesis, transport and secretion of bile acids.

Furthermore, organ weights of brain and heart were decreased in 1200 ppm females and thymus weights in 300 ppm males only. In contrast, liver weights were increased in 300 and 1200 ppm groups in both sexes. In line with clinical chemistry and organ weight findings, histopathology revealed hepatocytomegaly in 300 and 1200 ppm dosed males and females and changes in the livers of two dogs at 75 ppm. As the changes seen at 75 ppm were minor, seen in only one dog of each sex, and other evidence supports a physiologic response, the change at 75 ppm was not considered an adverse effect.

Based on these findings gallbladder and liver were determined as the primary target organs and an overall NOAEL was determined at 75 ppm (corresponding to 2.98 mg/kg bw/day (males) and 2.95 mg/kg bw/day (females)).

Rat

- chronic study, 2 years

In this GLP-conform 24 month combined chronic toxicity/carcinogenicity study conducted according to OECD 453, the test substance was administered in rodent diet at concentrations of 50, 100, 1000, and 3000 (male)/4000 (female) ppm to groups of 60 male and female Hsd/WIN: WU rats (1997f). Ten animals per group were selected for interim necropsy after 12 month. The mean test substance intake was 2.5, 10.3, 52.7 or 170.4 mg/kg bw/day for males and 3.6, 14.6, 75.4, 326.7 mg/kg bw/day for females.

Clinical observations were performed twice daily and once a day at weekends and bank holidays. Body weight was determined weekly from study start until Week 13 and every 2 weeks thereafter up to Week 105. Food and water consumption was determined and the average test substance intake was calculated. Opthalmoscopic examinations were performed 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 3000/4000 ppm dose groups were examined. At study termination 200 and 1000 ppm males were investigated, additionally. Blood collection for haematology and clinical chemistry was done at four time points (Weeks 26/27, 52, 78 and 103/104). Urinalysis was done in 10 animals per group six-monthly in Weeks 26/27, 52, 78 and 103/104. Additionally crystals in urine sediment were analysed in Week 103. Furthermore cholinesterase activity in the brain (Weeks 52/53 and 103/104/105), erythrocytes and plasma (Week 26/27) was determined. Weights of selected organs were determined at necropsy. Organs and tissues were subjected to gross pathological and histopathological investigations.

Weekly observations did not reveal abnormalities with regard to body surfaces and orifices, general behaviour, posture, respiration and excretory products up to the highest dose tested and there was no treatment-related effect observed on mortality and water intake. In 3000 ppm males and 4000 ppm females body weights were reduced and an increased incidence of skinny rats was noted at necropsy.

Food consumption was slightly elevated in 3000 ppm males and 4000 ppm females and together with the body weights indicated slightly reduced food efficiency in this dose groups. Males of the 1000 and 3000 ppm group had a higher incidence of moderate or severe opacities in the (whole) lens cortex. Since these alterations can be detected in rats of this age even at higher frequencies and differences to control values were very small and absent after 12 months, these findings are not considered an oculotoxic effect.

Haematological and histopathological investigations revealed no adverse effects on the red and white blood cells or the haematopoietic organs up to concentrations of 1000 ppm. Only in males of the 3000 ppm group slightly reduced haemoglobin concentrations and haematocrit values were measured in Week 26/27. These findings were attributed to the treatment, but since these effects were no longer visible at later time points, they are interpreted as marginal. In 4000 ppm females reduced MCH (Week 26/27) and MCHC (Weeks 26/27 and 52) values were observed. Furthermore elevated number of females showed increased pigment storage. Since effects on the primary parameters of the red blood were missing, these effects were not considered of toxicological relevance.

Clinical biochemistry revealed slightly, but sometimes significantly reduced aspartate aminotransferase and alkaline phosphatase activities and in 3000 ppm males and 4000 ppm females.

Determination of cholinesterase activity showed biologically relevant inhibition of the cholinesterase activity in erythrocytes from 1000 ppm onwards (both sexes). The brain cholinesterase activity was not affected in males up to 1000 ppm and females up to 200 ppm. At 4000 ppm females exhibited up to 41% lower mean cholinesterase activity in the brain compared with controls. The slight inhibition calculated in 1000 ppm females at interim (17%) and final necropsy (13%) as well as in 3000 ppm males (14%) at termination is considered to be of questionable toxicological relevance, since it was less than 20%

In the sciatic nerve of 3000/4000 ppm rats an increase in advanced degenerative myelinopathy was observed. Degenerative lesions of the skeletal muscles observed in 4000 ppm females, were considered secondary to this change.

In 3000/4000 ppm rats a vacuolation of cortical cells in the zona fasciculata of the adrenal glands occurred more frequently than in the other groups. Reduced spleen weights were observed in 3000 ppm males and 4000 ppm females, but there were no histopathological correlates to the reduced spleen weights noted. At 3000 ppm the relative testis weights were slightly elevated (about 17%). Since histopathological investigations revealed no morphological correlates this finding is considered to have no toxicological relevance.

There were no effects on the liver up to 200 ppm in males and females. At higher concentrations changes in liver function and morphology were observed. Cytoplasmic changes and/or hypertrophy of hepatocytes were found in 1000 and 4000 ppm females. Eosinophilic foci and cytoplasmic changes including focal degenerative changes were detected in 3000 ppm males. Single cell necrosis was noted in 4000 ppm females. A hepatocellular hypertrophy had been evident already in rats of the interim kill from 1000 ppm onwards. In 3000 ppm males and 4000 ppm females elevated liver weights (absolute and relative) at both necropsy time points and a slight increase in gamma-glutamyl transferase activity in the plasma were observed. There was no increase in hepatocellular tumours in treated rats.

In the thyroids follicular tumours (2 adenomas and 1 carcinoma vs. none in controls) and hyperplasias (7 vs. 1 in control animals) were noted in 3000 ppm males more often than in the other groups. Additionally, in 3000/4000 ppm rats a colloidal mineralization was detected more often. The observed incidences were within historical control data (adenomas up to 4.4%, carcinomas up to 2%) and the thyroidal findings can be interpreted as a consequence of altered thyroidal function by changes of the liver function, therefore treatment-related tumours observed are considered to have little relevance for humans under these circumstances.

In the urinary bladder of high dose males and females the incidence of focal or diffuse simple hyperplasia of the urothelium was significantly increased. An increase in proliferation rate of urinary bladder epithelium cells determined by proliferating cell nuclear antigen analysis was limited to 4000 ppm females (males were not investigated). A few transitional cell tumours in 4000 ppm females are considered to be sequelae to chronic irritation and, therefore, not a sign of a primary carcinogenicity. Changes in urine pH or microcrystals in the urine sediment were not observed. Since there were indications of a corrosive potential of two (cyclohexylamine and N-ethylcyclohexylamine) of four possible metabolites, an irritant or cytotoxic effect on the urinary bladder epithelium was postulated as being the trigger for the proliferations.

Altogether a proliferating potential on the urinary bladder epithelium due to a non-genotoxic mechanism was evident in males at 3000 ppm and females at 4000 ppm. The fact that at 200 and 1000 ppm two males of the interim kill group each showed a transitional cell bladder hyperplasia was not considered to reflect a compound effect for the following reasons: There was no increase in the proliferation index of bladder epithelium cells in 1000 ppm females, and none of the 200 and 1000 ppm males treated longer than 12 months exhibited pre-neoplastic urinary bladder lesions.

Furthermore, there are examples that one-year-old rats could develop a transitional cell hyperplasia spontaneously with a frequency of up to 20%.

Therefore, a neoplastic NOAEL level for the effects on the urinary bladder was established at 1000 ppm (corresponding to 52.7 mg/kg bw/day (males) and 75.4 mg/kg bw/day (females)). Uterine adenocarcinomas and one squamous cell carcinoma were observed in 4000 ppm females. However, the incidences of both tumour types did not exceed the historical control range and are therefore, considered to be not compound-induced.

In the other organs there was no evidence of treatment-related alterations, no shift in the tumour spectrum and no increase in the total number of tumours or the incidence of animals with tumours which can be attributed to the treatment with the test substance. Based on these findings a non-neoplastic NOAEL of 200 ppm (corresponding to 10.3 mg/kg bw/day (males) and 14.6 mg/kg bw/day (females)) was determined for male and female rats.

- sub-chronic study, 90 days

A sub-chronic 13 week study in Wistar rats with a 4 week recovery period to determine reversibility of possible effects was performed GLP conform and according to OECD 408 (1997u). Ten animals per sex and treatment level received a diet with test substance concentrations of 20, 100, 400, 1600 and 6400 ppm. The average daily test substance intake was 1.5, 7.2, 29.8, 136.6 and 660.9 mg/kg bw/day in males and 1.9, 8.8, 35.5, 174.1 and 700.8 mg/kg bw/day in females. Controls received plain diet (with 1% peanut oil to avoid dust formation) only. The additional recovery groups consisted of control animals and animals dosed at 6400 ppm.

Examinations included occurrence of mortality, clinical signs, changes in bod weight and weight changes, food and water consumption and test substance intake, haematological and clinical biochemistry analysis, urinalysis, ophthalmoscopic examinations, gross pathology examinations, determination of organ weights and histopathology analysis. In addition liver enzymes were determined and electron microscopic examinations of urinary bladders were performed.

General behaviour and clinical findings did not differ between treated and control animals and no mortalities or ophthalmoscopic effects were observed.

Significant reduced body weights were observed in males at doses of 1600 ppm and above and in females in the highest dose group. At the end of recovery period a trend to reversibility of the effects on body weight was visible.

Toxicological relevant changes in feed or water consumption and test substance intake were not observed. Resulting from the body weight depression the intake data per body weight were increased in the 1600 ppm dose group and/or 6400 ppm group.

Haematology analysis revealed reduced haemoglobin and haematocrit values as well as an increase in Met-haemoglobin content and reticulocytes count in animals dosed at 1600 ppm and above. In addition, coagulation time was shorter in females of the highest dose group. A toxic influence of the compound on the red blood cells in males (1600 ppm and above) and females (6400 ppm) was assumed. However, at the end of the recovery period, haematological measurements did not reveal any toxicologically relevant changes in red or white blood parameters.

Cholinesterase activity in erythrocytes was significantly and biologically relevant inhibited (more than 20%) in animals dosed at 1600 and 6400 ppm.In the plasma cholinesterase activity was only slightly reduced in the highest dose group. Comparable lesions were absent in rats of the 6400 ppm recovery group. Observed effects on cholinesterase activity were partially reversible.

At concentrations above 400 ppm elevated cholesterol means, reduced triglyceride concentrations and decreased contents of unesterified fatty acids (at 1600 and 6400 ppm) as well as decreased albumin and increased urea levels (6400 ppm) in the peripheral blood were observed, indicating changes in liver function. Furthermore, an induction of liver enzymes, gamma-glutamyl transferase (plasma) and O-demethylase (liver) was observed. In line with these findings histopathology revealed hypertrophic hepatocytes with a honeycombed cytoplasmic structure in rats receiving 1600 and 6400 ppm. In addition, statistically significant elevated liver weights were observed in 6400 ppm males (relative: 37%) as well as in 1600 ppm (relative: 12%) and 6400 ppm (absolute; 35%; relative: 44%) females. Taken together these data point at the liver as a primary target organ. However, observed liver effects turned out to be reversible.

In the highest dose diffuse simple hyperplasia of the bladder urothelium accompanied with small round cell infiltrates was evident. The additional electron microscopic examination of urinary bladders confirmed cytotoxicity of the test substance on the superficial layer of the urothelium of the urinary bladder in male Wistar rats with consequent mild regenerative hyperplasia. Results of the recovery group showed that these findings were reversible.

Higher relative testes weights observed in the 6400 ppm group were not considered a toxicologically relevant effect, since histopathology revealed no alterations and might be related to reductions in body weight.

Taken together urinalysis and the lack of kidney alterations in gross pathology and histopathology examinations, there were no indications for kidney damage up to a dose of 6400 ppm.

An increase in vacuoles in the adrenal zona fasciculata and the reduction of thyroid follicle size in rats dosed at 1600 and 6400 ppm were considered secondary possibly due to the effect on the liver. Thyroid follicles were reduced in size in males receiving 1600 and 6400 ppm as well as in 6400 ppm females. Additionally, they exhibited a lumpy colloid. Except of the lumpy colloid of females these alterations were reversible. Since none of the thyroid hormone parameters (T3 and T4) were changed, the morphological alterations were considered to reflect unspecific changes in thyroidal physiology. Effects on adrenals and thyroids were considered potentially secondary to reactions due to alterations in the liver.

In this rat study the test substance was tolerated without adverse effects at doses of up to 400 ppm. The NOAEL was therefore 400 ppm, corresponding to a mean test substance intake of 29.8 and 35.5 mg/kg bw/day test in males and females, respectively.

- sub-acute study, 28 days

A sub-acute 4 week study with a 2 week recovery period was performed in Wistar rats similar to OECD guideline 407 (1998b). The test substance solved in olive oil was administered per diet. Six animals per sex and dose received dietary concentrations of 100, 1000 and 10000 ppm. The average daily test substance intake was 9.17, 105.6 and 1089 mg/kg bw/day in males and 10.54, 103.6 and 1083 mg/kg bw/day in females. The additional recovery groups consisted of control animals and animals dosed at 10000 ppm. Controls received diet with 0.8% olive oil only. Examinations included clinical signs, determination of body weight, food and test substance intake, food efficiency, haematological and clinical biochemistry analysis and urinalysis. At necropsy gross pathology examinations, determination of organ weights and histopathology analysis were performed.

Treatment-related clinical findings and deaths were not observed up to 10000 ppm, but at the highest dose tested body weights were decreased in both sexes, a transient decrease in food consumption was observed at the beginning of administration and food efficiency showed a decreasing tendency. In the recovery group, differences in body weight or body weight gain were only visible in males and food efficiency showed an increasing tendency.

Haematology revealed a significant decrease in haemoglobin, corpuscular indices and a tendency of decreased haematocrit values in both sexes. In addition females had significantly decreased white blood cell count. With exception of the decreased haemoglobin values, effects were reversible. Since morphological alterations were not detected in haematopoietic organs, the effects were considered secondary. But differential count of leukocytes (%) in dose groups did not differ from that of controls.

Analysis of clinical biochemistry parameter showed increased blood urea nitrogen levels in males at 1000 ppm and above. Furthermore, in the highest dose group kidney weights were reduced in males and females. But histopathology and urinalysis provided no signs indicating effects on kidney function.

Clinical biochemistry also revealed an increase of total cholesterol in males at 1000 ppm and above and in females at 10000 ppm. This finding may be associated with alterations of the lipid metabolism, although the triglyceride level was unaffected. At 10000 ppm also a decrease in cholinesterase activity in blood serum was visible in both sexes No data on ChE activity in erythrocytes is available and thus the effect in serum is not considered toxicologically relevant according to ECHA 2016.Further findings were an increase of chloride levels in males treated at 100 ppm and above and decreased calcium levels in females receiving diet with 100 and 1000 ppm test substance. Since a dose dependency was not observe the decreased calcium levels were not considered of toxicological relevance.

Taken together clinical biochemistry, gross pathology and histopathology data, the liver was identified as a primary target organ. Glutamat-oxalacetat-transaminase levels were significantly decreased in both sexes and glutamat-pyruvat-transaminase in males. Furthermore, albumin and albumin/globulin ratio was decreased at 10000 ppm. In line with these findings dark colouration of the liver was observed in both sexes and absolute and relative liver weights were increased at 10000 ppm. In females increased relative liver weights were already visible at a dose of 1000 ppm. Histopathology revealed irregular distribution of eosinophilic cytoplasm in hepatocytes in males dosed at 100 ppm and above. In addition, swelling of hepatocytes was observed in both sexes at 100 and 10000 ppm. In females this finding was accompanied by eosinophilic bodies in hepatocytes and local granulation in the liver. Liver effects were partially reversible. In males, the findings for eosinophilic body and swelling of hepatocytes disappeared and dark colouration was only observed in one male at the end of recovery period. Whereas liver weights in females were still increased.

Histopathology also revealed vacuolization of adrenal cortex in females at 1000 ppm and above and in males at 10000 ppm, immature thyroid glands in females at 1000 ppm and above and in males at 10000 ppm, extramedullary haematopoiesis in spleen of males and vacuolization of bone marrow in females of the highest dose group. Furthermore, increased relative brain and testes weights were observed in males of the highest dose group, whereas absolute spleen weights in males and absolute adrenal weights in females were decreased.

Based on the observed findings the liver was identified as primary target organ. Other effects on thyroidal glands, adrenal glands and cholinesterase inhibition were considered to need further examinations in longer toxicity studies. A NOAEL was therefore estimated at 100 ppm for both sexes, corresponding to 9.17 mg/kg bw/day in males and 10.54 mg/kg bw/day in females.

- sub-acute study, 7 days

A 7-day sub-acute toxicity study in 5 rats (Crj:CD (SD)) per sex and dose was performed (1994a). In this study the test substance suspended in 2% Cremophor EL DW was administered per gavage at 2500 mg/kg bw/day for seven days. Examinations included clinical findings, observation of mortalities, body weight, haematology and clinical biochemistry analysis, gross pathology examinations, organ weights and histopathology examinations.

No deaths, clinical findings or effects on body weights were observed. At 2500 mg/kg bw/day increased white and red blood cell counts were observed in males, decreased haemoglobin and haematocrit values were visible in males and females, mean corpuscular haemoglobin was decreased in males, mean corpuscular volume was decreased in females and reticulocytes were increased in males.

Clinical biochemistry analysis revealed significantly decreased aspartate aminotransferase in males, significantly increased creatinine levels in males, significantly increased blood urea nitrogen in females, significantly increased total cholesterol and appearance of Heinz bodies in both sexes.

Kidneys were pale and kidney weights slightly but not statistically significantly increased in females. Furthermore, in the kidneys of treated females calcificated cortico-medullary regions were observed and in kidneys of males eosinophilic tubules. Livers were enlarged and weight increased in treated animals. In addition, histopathology revealed extramedullary haematopoiesis in the liver of males and females, indicating a treatment-related adverse effect on the liver. Measurement of organ weights also revealed decreased thymus weights in males, however histopathological correlates were not observed.

Because only one dose was investigated, dose-dependency could not be evaluated in this study. Without historical control data and because of very limited documentation it was not possible to conclude for all observed effects whether effects were treatment-related or incidental. A NOAEL was not determinable.

Mechanistic studies

- rats, myelinopathies

A special sub-acute study in compliance with GLP was performed in which the test substance was administered to rats in their diet for 2 weeks (1998e). The study was conducted to gain further insight in the pathomechanism of increased incidences of degenerative myelinopathies observed in the chronic rat study (1997f).

Therefore, focus of the study was to evaluate the plasma concentration of the test substance and its main metabolite chlorophenyl-tetrazolinone (CPT). In addition, activity of neuropathy target esterase (NTE) in brain, spinal cord and sciatic nerves as well as brain cholinesterase (ChE) activity were measured. Furthermore, urinary sodium and urinary pH were measured to evaluate a possible impact of these parameters on the urinary bladder epithelial hyperplasia observed in the chronic rat study.

Rats (5 per sex/dose level) received dietary concentrations of 50, 200, 1000 and 4000 ppm test substance. Two additional groups of 5 male rats each were included as vehicle and positive control group (500 mg/kg tricresyl phosphate (TOCP)) for ascertaining the well-known inhibition of NTE in rodents (satellite groups). Control substances were administered once on Day 0 in a single dose by stomach tube. NTE activity was assayed in brain, spinal cord and sciatic nerves excised 24 h after dosing. Plasma concentration of the test substance and its main metabolite CPT were examined in all animals at term of study as well as inhibition of NTE in brain, spinal cord and sciatic nerves and brain ChE activity. Urinary pH and urinary sodium ion concentration were examined after 8 days (male) or 9 days (female) of treatment.

Survival rate and general behaviour of the animals were not affected by the treatment with the test substance. Mean food consumption was increased in both sexes at 4000 ppm, but there was no effect on body weight development. Up to 1000 ppm plasma concentrations of the test substance were below the limit of detection (0.5 nmol/mL) and in the 4000 ppm dose group near the limit of quantification (1 nmol/mL). Only in 3/5 male animals and 4/5 female animals of the 4000 ppm group the test substance signal could be detected. The main metabolite CPT could be detected at dietary levels of 200 ppm and above in both sexes. The concentration of CPT increased dose-dependent but not dose-proportional up to the 4000 ppm dose groups and reached mean values of 9.8 nmol/mL (males) and 13.9 nmol/mL (females) in the high dose group.

The positive control, TOCP, strongly inhibited NTE one day after administration in male rats by almost 90% in brain and spinal cord, and by 77% in sciatic nerves, thus demonstrating the sensitivity of the test system. Following 2 week administration of the test substance to male rats generally no NTE inhibition was observed. Only in brain of the highest dose group, a statistically significant NTE inhibition of 13% was found. Likewise, female rats exhibited mild, statistically significant NTE inhibition only in the highest dose group in brain (14%), spinal cord (15%) and in sciatic nerves (29%). These mild NTE inhibitions demonstrate the absence of a potential of the test substance to induce nerve degenerations similar to an organophosphate induced delayed neuropathy. Brain ChE activity was equivocally inhibited in 4000 ppm males and in 1000 ppm females and was significantly inhibited in 4000 ppm females. These results were comparable to that observed in the chronic study.

Examination of urinary pH and urinary sodium ion concentration did not reveal significant differences in mean urinary pH or urinary sodium ion concentration. Marginally decreased urinary sodium ion concentrations in male rats at doses of 1000 and 4000 ppm were not considered treatment-related since this difference was not confirmed in females.

In conclusion, an overall NOEL of 1000 ppm with regard to NTE inhibition was determined. The overall NOEL for ChE inhibition was 1000 ppm for males and 200 ppm for females.

- rats, effects on erythrocytes (in-vitro)

The effect of the test substance on rat erythrocytes was tested in a GLP compliant in-vitro study (2000f). Aim of the study was to clarify the mechanism of the effects the test substance on red blood cells previously observed in rats in-vivo (please refer to Chapter 1.3). Therefore the test substance and its metabolite o-chlorphenyltetrazolinone (CPT) dissolved in DMSO were tested at concentrations of 0.1, 1, 5, 10, and 50 µg/mL in rat erythrocytes in-vitro for seven days. Evaluations were made at Day 1, 3 and 7 after the first dosing and examined parameters included haemolysis, intracellular ATP and GSH level, glucose consumption, and met-haemoglobin formation.

One day after treatment glucose utilization of erythrocytes was concentration-dependently depressed as demonstrated by strong inhibition of glucose consumption and to a smaller extent, of intracellular ATP content. Also GSH content was strongly decreased indicating a breakdown of this important protective system in test substance-treated erythrocytes. Only marginal haemolysis was observed at Day 1, whereas marked haemolysis was observed at Days 3 and 7 (NOEC 1 µg/mL at Day 7). At Day 3 and 7, ATP content was significantly decreased and the most sensitive parameter with a NOEC of < 0.1 µg/ml. Treatment with the metabolite CPT up to a concentration of 50 µg/mL had no effect on any of the test parameters.

It was concluded that the test substance at concentrations relevant for the high doses of the sub-chronic and chronic rat studies impairs the glucose utilization of the erythrocytes via inhibition of glycolysis. This compromises the integrity of the circulating mature erythrocytes which results in a slight haemolytic anaemia with compensatory reticulocytosis. Due to the low bioavailability of the test substance in rats the effects observed in-vivo were only marginal and occurred only at high doses.

- hogs, effects on thyroid peroxidase (TPO) (in-vitro)

In-vitro studies on the interaction of the test substance with thyroid-peroxidase-catalyzed reactions were performed in compliance with GLP (1997v).

Reasons for conduction of these studies were effects observed in the chronic rat study. In this study, slightly increased incidences of colloid mineralization (both sexes), follicular hyperplasia and adenomas (males only) were observed at the highest dose level. Besides secondary effects, a direct inhibitory effect of the test substance on thyroid peroxidase (TPO) was suggested as the underlying mechanism.

Therefore, to investigate a potential direct inhibitory effect of the test substance on TPO, interactions with TPO-catalyzed reactions were studied in-vitro using solubilized hog thyroid microsomes as an enzyme source. Examinations included determination of the peroxidative activity, measured by oxidation of the model substrate guaiacol, and determination of TPO-catalyzed iodine formation.

Up to the highest applicable concentration of 100 µM, the test substance did not inhibit TPO-catalyzed oxidation of the model substrate guaiacol. In contrast, amitrole, which was used as positive control substance, strongly and concentration-dependently inhibited guaiacol oxidation. In the test with the physiological substrate iodine, treatment with the test substance at 25 µM did not affect TPO-catalyzed iodine formation from iodide. Of note is that when iodide was used as a substrate instead of guaiacol, the solubility of the test substance was very limited, resulting in reduced maximal concentration. It was suggested that the presence of guaiacol had improved the solubility of the test substance. The positive control effectively and concentration-dependently inhibited the initial rate of this reaction.

These findings suggest that the test substance neither inhibits the enzyme itself, nor is able to trap the iodinating species generated by TPO. Thus, a direct inhibitory effect of the test substance on thyroid peroxidase was considered unlikely.

- mice, rats, rabbits, effect on physiological functions

The effect of the test substance on physiological function was testes in mice (ICR), rats (Sprague Dawley) and rabbits (Japanese white strain) (1997w).

The aims of the present study were to investigate general pharmacological properties and to clarify potential toxicity of the test substance.

The test substance was administered orally at a dose of 1000 and 5000 mg/kg bw to mice/rats or 500 and 2500 mg/kg bw to rabbits, to test its influence on general behaviour, effects on the central nervous system (mice and rabbits), the autonomic nervous system (rabbits), the influence on respiration, blood pressure, heart rate and electrocardiogram (rabbits), the influence on the somatic nervous system (motor coordination and muscle force; mice), the influence on the digestive system (charcoal transit in the gastro-intestinal tract; mice), the influence on the renal function (rats), the influence on blood coagulation and haemolysis (rats), the influence on hypo-osmotic pressure-induced haemolysis (rats). In addition the influence of the test substance on plasma cholinesterase (ChE) activity was tested in male rabbits at a dose of 500, 1000, 2500 and 5000 mg/kg bw.

The general state of mice and rabbits were not changed by the test substance at both respective dose levels. Also spontaneous movement of mice was not changed by the test substance at any administered dose. Rectal temperature and pupil size of rabbits were not changed by the test substance at both doses. In addition, the respiratory and circulatory systems in conscious rabbits were not markedly affected by the test substance.

Motor coordination in mice tested using a rotating rod apparatus and traction force of the skeletal muscles tested with suspension-wire were not affected after treatment with the test substances at both doses. Charcoal transit through the gastro-intestinal tract of mice was not affected by the test substance at any tested dose.

The urinary volume and pH, and excretions of sodium, chloride and potassium showed no remarkable changes in rats treated with the test substance 6 h after administration. In detail at a dose of 1000 mg/kg bw, significantly increased K+ excretion was observed in urine. However, urinary volume, and excretions of Na+ and Cl- did not change. Although the increase in K+ excretion was significant, the increase was very slight. At a dose of 5000 mg/kg bw, decreased urinary volume, Na+ and Cl- excretion and slightly increased K+ excretion was observed. However these changes were not significant. Urinary pH was not affected by the test substance at both doses. Therefore, it was suggested that the changes in renal function of rats is not necessarily treatment-related. Analysis of prothrombin time and partially activated thromboplastin time were not changed in blood samples obtained from rats at both dose levels. Also osmotic resistance of rat erythrocyte to hypo-osmotic challenge was not changed.

In rabbits, plasma ChE activity was decreased at all doses. The test substance significantly decreased the ChE activity 1 day after administration of 500 mg/kg and from 2 h to 1 day after administration of 2500 mg/kg. At a dose of 1000 mg/kg plasma ChE activity was decreased 6 h and 1 day after test substance administration. At a dose of 5000 mg/kg, the activity was decreased starting 2 h after administration. Furthermore 2/3 rabbits treated at the highest dose were found dead at the next morning after treatment.

Since the test substance did not affect the general state, the pupil size, blood pressure, heart rate, respiration and EGG in conscious rabbits, it was suggested that the test substance does not cause symptoms like intoxication of ChE inhibitor, though it decreased the activity of the enzyme in plasma. Actually no toxic effects, which were supposed to be due to the inhibition of ChE, were observed in mice, rats and rabbits, though two rabbits died after dosing.

In a previous conducted study in rats ChE activity in plasma, brain and erythrocyte was decreased in rats at a dose of 5000 mg/kg bw. In contrast to this study, where deaths were observed at this dose level in rabbits, no deaths were observed in the previous study in rats, indicating species differences in the sensitivity to the test substance.

Taken together all results it was demonstrated that the test substance has no marked effects on the general state, behaviour, the central nervous system, the autonomic nervous system, the respiratory and circulating system, the somatic nervous system, the digestive system, the renal function and on blood or blood cells, though the test substance markedly decreased the ChE activity in rabbits plasma. In addition, it is also indicated that the test substance at the doses up to 5000 mg/kg bw in mice and rats, or to 2500 mg/kg bw in rabbits, caused no acute toxic symptoms.

- mice/dog, immunochemistry for gallbladder effects

A sub-chronic feeding study in female mice and dogs were performed for characterization of gallbladder effects (2000b). In this study, the test substance was administered in feed at dose levels of 20, 100, 600, 3600 and 7200 ppm to 10 mice per dose level and at 75, 300 and 1200 ppm to 4 dogs per dose level. Tissue sections of the gallbladder and liver were obtained from the original paraffin blocks used in the sub-chronic feeding study in mice (1997h) and dogs (1996c). The proliferating mucosal epithelial cells were identified by immunohistochemistry for proliferating cell nuclear antigen (PCNA). The gallbladder and liver sections were stained with abidin-biotin complex (ABC) method using an anti-PCNA antibody (anti-PCNA, PCIO mouse monoclonal antibody). As a positive control, PCNA immunoreactivity was monitored on liver sections. PCNA-stained cells were quantified by microscopic analysis. Positive (S phase cells) and negative mucosal epithelial cells were counted until a total of 400 cells for mice or 1000 cells for dogs were achieved. Labelling indices of PCNA (number of S phase cells/total number of cells counted x 100) were determined in gallbladder sections of each animal. Liver sections were not subjected to S phase cell counting.

In mice a significant increase in PCNA labelling indices in mucosal epithelial cells of the gallbladder were observed in animals receiving 7200 ppm. At the next lower dose level, 3600 ppm, an increasing trend in PCNA labelling indices was visible. At all other dose levels no significant effects were observed. The results were considered to be treatment-related because they were dose-related. In dogs no significant changes in PCNA labelling indices of the gallbladder were observed at any dose level. The positive control tissue (liver) sections were positive for all mice and dogs. It was concluded that the test substance may enhance cell proliferation activity in the mucosal epithelial cells of the gallbladder in female mice following sub-chronic administration of the test substance at 3600 and 7200 ppm.

- mice, liver enzyme activity, protoporphyrin IX in liver and bile, bile fluid analyses

A special mechanistic GLP conform study in mice was performed to investigate biochemical liver and bile fluid parameters (1997i). In this study the test substance was administered to 80 female B6C3F1 mice in their diet at concentrations of 10000 ppm for up to 8 weeks.

Bases for these investigations were changes in liver function, dark-colouration of bile fluid and hyperplastic changes in the gall bladder urothelium observed in the sub-chronic study in mice (1997h). Since liver and bile protoporphyrin IX plays an important role in the biliary excretion of pigmented metabolism products, analysis of porphyrin biosynthesis was performed. In addition, to get insight into possible reasons for discoloured gallbladder material, the cholesterol content, the pattern of bile acids and the occurrence of the test substance and its metabolite o-chlorophenyl-tetrazolinone (CPT) in the bile fluid were analysed.

No clinical symptoms or mortalities were observed in mice treated with the test substance. At necropsy the gallbladder of treated mice contained a dark coloured and partly solid material. Analysis of porphyrin biosynthesis did not reveal any effect in treated mice. The solid material did not contain remarkable concentration of cholesterol. In addition, no significant amounts of the test substance and its metabolite CPT were observed in the bile fluid, indicating that there is no relevant biliary excretion of these compounds in mice. Analysis of the hepatic enzyme pattern revealed a strong induction of cytochrome P450-dependent monooxygenases and a slight induction of Phase II enzymes in the treatment group. 1H-NMR spectroscopy of the gallbladder content revealed an elevated bile acid synthesis indicated by a higher concentration of taurocholic acid. This bile acid amounted to 40% of the bile material. It was concluded that the test substance is a strong cytochrome P450-inducer in mice, which is different to the situation in rats. This effect is postulated to trigger enhanced bile acid synthesis.

- dogs, liver enzyme and gall bladder toxicodynamics

A mechanistic GLP-conform study on liver enzyme and gall bladder toxicodynamics in Beagle dogs fed with the test substance was conducted (1997g).

Purpose of the study was to characterize the mechanism of induction and recovery of microsomal liver enzymes and mechanism of gall bladder changes. One set of 4 control and 4 treated female Beagle dogs were fed diet at a nominal test substance concentration of 750 ppm, corresponding to a test substance uptake of 30.6 mg/kg bw/day, for 46 days. The other set of 4 control and 4 treated female Beagle dogs were fed diet at a nominal test substance concentration of 750 ppm, corresponding to a test substance uptake of 31.9 mg/kg bw/day, for 42 days followed by a recovery period of 46 days during which the dogs received control diet.

Examinations included determination of body weight and food consumption, clinical observations, and clinical chemistry analysis (pre-treatment, 2 and 6 weeks of treatment). In addition, the following liver enzymes (Phase I enzymes: N-, O-demethylase, cytochrome P-450 and uridine diphosphate glucuronosyltransferase) were analysed in liver samples of all dogs at necropsy. For the experiment with recovery phase the following Phase I and II liver enzymes were measured in liver samples taken from the control and treated group at the end of the treatment and recovery phase: ethoxycoumarin o-deethylase, ethoxyresorufin dealkylase, aldrine epoxidase, epoxide hydrolase, glutathione-S-transferase, uridine diphosphate glucuronosyltransferase, cholesterol 7 hydroxylase (CYP7A). At study termination gross pathology and light microscopic examinations of the liver and gall bladder were performed.

A test substance-related decrease in food consumption was observed in the 750 ppm treatment group. The food consumption per body weight was slightly decreased in the treatment phase (Days 1-46), and the first 20 days of the recovery period (Days 47-67), but was slightly increased during the last 20 days of the recovery period (Days 68-88) in the treated group compared to the corresponding controls. There was also a compound-related decrease in body weight in the treatment group compared to controls. In the treated recovery group a trend of increased relative body weight was visible. No relevant clinical signs that were attributed to the test substance were observed.

After the treatment phase of 46 days, O- and N-demethylase and cytochrome P-450 were significantly increased in hepatic tissue. In addition, hepatic cytochrome P-450-dependent monooxygenases (ethoxycoumarin o-deethylase and aldrine epoxydase) and epoxide hydrolase were significantly increased compared to concurrent control values, while ethoxyresorufin dealkylase was significantly decreased from controls. The conjugation enzyme, UDP-glucuronyltransferase (UDP-GLU-T), the rate limiting enzyme in thyroxine metabolism was statistically increased after treatment.

Both UDP-GLU-T levels, along with other hepatic enzymes reversed to normal levels in the recovery animals. Both O- and N-demethylase had decreased to significantly lower than concurrent control values at the terminal sampling, though the difference was slight. It was reported that CYP7A, the first and rate limiting step in the conversion of cholesterol to bile acids, was significantly increased after treatment and also fully recovered to control levels.

Mean bile acids (in bile) were slightly decreased from control values in treated dogs after the treatment phase, and slightly increased over concurrent controls after the recovery phase, but most likely reflected changes in biliary flow and acid production during treatment and adaptive changes during recovery. None of the data were significantly different from concurrent controls.

Micropathology analysis revealed a "ground glass" appearance of hepatocellular cytoplasm consistent with proliferation of endoplasmic reticulum (4/4 animals), and submucosal oedema of the gall bladder (3/4 animals) in the treatment group. In the recovery group the hepatic changes and gall bladder oedema had resolved almost entirely, but a slight cholecystic mucosal hyperplasia (2/4 animals) was present. Relative, liver weights were significantly increased in the 750 ppm group after both treatment and recovery phase. Absolute liver weights did not differ between groups.

The mechanistic feeding study demonstrated reversibility of the test substance-induced hepatic enzymes N- and O-demethylase and cytochrome P-450, as well as other hepatic cytochrome P-450 dependent monooxygenases. The conjugation enzyme, UDP-GLU-T was statistically increased after treatment, thus substantiating the conclusion of a hepatic extra-thyroidal mechanism in the sub-chronic dog study. Changes in bile acid production were suggested to contribute to gall bladder mucosal hyperplasia observed in this and other studies. It was concluded that the liver enzyme induction effects have been proved to be reversible and not an adverse toxicological effect, as previously reported in the chronic (20 ppm NOAEL) and sub-chronic (75 ppm NOAEL) dog studies.

- dogs, liver enzyme activities

A mechanistic sub-chronic feeding study in dogs to investigate the induction of liver enzyme activities, including cholesterol 7 alpha- hydroxylase (CYP7A) was performed in compliance with GLP (1998d). Reason for this study were indications from other sub-acute and sub-chronic studies in mice and dogs showing a strong induction of microsomal liver enzymes (Phase I, cytochrome P-450's) with hints for an increasing biosynthesis of bile acids in the mouse (gallbladder) and dog (bile acids in plasma). From published literature, there were indications that there may be a connection between the induction of cytochrome P-450 enzymes and bile acid synthesis. Different literature sources show that cytochrome P-450 CYP7A catalysed the first and rate limiting step in the conversion of cholesterol to bile acids in mammalian liver [1, 2, 3]. Therefore, the aim of this mechanistic dog study was to reveal the induction of different cytochrome P-450 enzymes inclusive CYP7A and the induction of Phase II enzymes with a particular interest in the UDP-glucuronyl transferase in the liver.

In this study, the test substance was administered in the diet at a concentration of 750 ppm to 4 female Beagle dogs for a period of 46 days. An additional group was treated with the test substance for 42 days followed by a 6 week recovery period, to examine the normalization of enzyme activities. After 46 or 42 Days of treatment (+ 6 weeks of recovery), liver tissues were examined for the activities of cytochrome P-450 dependent monooxygenases (hepatic CYP7A, 7-ethoxycoumarin deethylase (ECOD), 7-ethoxyresorufin deethylase (EROD), aldrin epoxidase (ALD), epoxide hydrolase (EH)) and the conjugation enzymes (glutathione-S-transferase (GS-T), UDP-glucuronyl transferase (UDP-GLU-T)).

Analysis after a treatment period of 46 Days revealed that the test substance induced monooxygenases ECOD (8 fold) and ALD (2.8 fold). In contrast EROD (CYP1A1) was reduced to about 20% of the control group. In addition, a significant induction of the EH (2.5 fold) and of the UDP-GLU-T (2.4 fold) was observed. GS-T was not enhanced. After the recovery period all measured enzyme activities were similar to that of controls without any significant differences. The mean hepatic CYP7A activity was significantly increased in dogs receiving 750 ppm test substance compared to controls (1.9 fold higher).

It was concluded that the effect of the test substance plays an important role for some secondary findings observed in dogs. It was assumed that the induction of different cytochrome P-450 subtypes inclusive CYP7A could be responsible for the decreased serum cholesterol level in the sub-chronic study and for the hyperplasia in the gall bladder by a reflection of increased bile acid synthesis. In addition, it was assumed that the increased activity of the UDP- GLU-T could be responsible for decreased thyroxine serum levels in the sub-chronic dog study.

References:

[1] Pandak, W., Vlahcevic, Z., Chiang, J., Heumann, D., Hylemon, P., 1992 Bile-acid synthesis: Regulation of Cholesterol 7-alpha-hydroxylase by Taurocholate and Mevalonate, J. Lipid Res. 33(5), 659-668

[2] Shefer, S., Kren, B. T., Salen, G., Steer, C. J., Nguyen, L. B., Chen, T., Tint, G. S., Batta, A. K., 1995 Regulation of bile acid synthesis by deoxycholic acid in the rat: Different effects on cholesterol 7α‐hydroxylase and sterol 27‐hydroxylase, Hepatology 22(4), 1215-1221

[3] Waxman, D. J., 1986 Rat hepatic cholesterol 7α-hydroxylase: biochemical properties and comparison to constitutive and xenobiotic-inducible cytochrome P-450 enzymes, Arch. biochem. biophys. 247(2), 335-345

- dogs, effects on bile flow and composition

A mechanistic GLP-conform study on bile flow, bile composition and liver histology in chronically bile duct-cannulated female beagle dogs fed with the test substance (750 ppm) for up to 6 weeks was conducted, to elucidate the pathogenesis of gall bladder mucosal hyperplasia observed in previous dog studies (2000a).

Bile-duct canniculation started before administration of the test substance that is why dogs served as their own pre-treatment controls for bile flow and bile composition. Bile flow was recorded daily. The bile composition was analysed once a week. Further examinations included animal appearance, behaviour, food intake, body weight, haematological and clinical chemistry analysis and histopathological investigations of liver and changed tissue.

Treatment-related effects on behaviour, body weight and food consumption were not observed. However, several clinical disorders were related to the experimental conditions of the animal model (vomiting, hysterical attack, icterus and peritonitis due to displaced bile duct catheter, bacterial sepsis due to ascending infection along the catheters, leakage of the bile delivering catheter). Two dogs had to be sacrificed prior to scheduled necropsy. Haematology did not reveal any test substance-related changes, but the following liver specific parameters were increased: alanine aminotransferase, alkaline phosphatase and glutamate dehydrogenase activities. Both stress of the liver under chronic bile duct cannulation as well as treatment with the test substance could be responsible for these changes.

Gross and histopathology revealed gall bladder atrophy and residues of blood and bile in the gall bladder, which were considered related to surgical manipulations and/or a consequence of cystic duct ligation. Test substance-related effects on the gall bladder were not observed. Gall bladder mucosa hyperplasia observed in previous studies were therefore not considered a systemic, but a local effect which did not occur in the present study in which the gall bladder was surgically separated from the bile stream.

Another histopathology observation, cytoplasmatic changes of hepatocytes, was considered treatment-related and correlated to induced microsomal enzyme activity and with previously determined CYP7A induction.

Bile and bile flow analysis revealed a treatment-related change in bile colour (from dark green to yellow-brown), starting after 2, 3 or 4 weeks of administration. In addition, a marginally to clearly increased bile flow (13-63% above the respective pre-treatment flow) was determined in 3/4 dogs.

Analysis of bile contents revealed only low concentrations of the test substance and its metabolites. The concentration of cholesterol in bile (a material known to form gall bladder concretions) decreased during treatment and is therefore unlikely to be involved in the pathomechanism of gall bladder hyperplasia. However, bile acid synthesis was changed. Concentrations of the taurine conjugated bile acids taurocholic acid and taurodesoxycholic acid were decreased, whereas non-conjugated bile acids like cholic acid and desoxycholic acid and also other, non-identified bile acids increased significantly or appeared new in the bile during treatment with the test substance. Non-conjugated bile acids were assumed to have a higher irritating potential than conjugated bile acids, especially, when concentrated in the gall bladder. Therefore, the shift in bile acid composition towards a higher concentration of non-conjugated bile acids was considered most likely causally related to the gall bladder mucosa hyperplasia observed in previous studies.

CYP7A, is the rate limiting enzyme in the conversion of cholesterol to bile acid. In a previous study it was observed that CYP7A activity and bile acid biosynthesis were enhanced, which possibly exhausted the taurine conjugation capacity of the liver.

- rats, liver enzyme activity, protoporphyrin IX in liver and ChE activities

This 4-week repeated dose toxicity study in Wistar rats was performed in compliance with GLP for determination of liver enzyme activities and protoporphyrin IX in the liver, which plays an important role in the biliary excretion of pigmented metabolism products; and measurement of cholinesterase (ChE) activity in plasma, serum and brain (1997j).

The test substance was administered to 5 rats per sex and dose in their food at 10000 ppm for 4 weeks. The mean test substance intake was 903.9 mg/kg bw/day in males and 1357.8 mg/kg bw/day in females, respectively.

Examinations included clinical observations, mortalities, measurement of body weights, determination of food and test substance intake, ChE activity in plasma, erythrocytes and brain. In addition, biochemical liver parameters including cytochrome P450-dependent monooxygenases (7-ethoxycoumarin deethylase (ECOD), 7-ethoxyresorufin deethylase (EROD) and aldrin epoxidase (ALD)) and the Phase II enzymes (epoxide hydrolase (EH), glutathione-S-transferase (GS-T) and UDP-glucuronyl transferase (UDP-GLU-T)) were analysed in all animals. Furthermore, protoporphyrin IX was determined in liver tissue in one control male, two 10000 ppm males, and one 10000 ppm female. Gross or histopathology analyses were not performed.

Treatment with the test substance did not induce mortalities and no clinical findings were observed. In rats receiving 10000 ppm test substance body weights were significantly reduced by about 8-10% in males and females and the food uptake was markedly increased in males by 38% and females by 65% compared to controls.

Following treatment slight changes in the activity of monooxygenases but a pronounced induction of Phase II enzymes was observed. In treated males, the monooxygenase ECOD increased 1.5 fold, but the EROD and ALD activities were decreased up to 50% compared controls. EH increased 4 fold, the GS-T 3 fold and the UDP-GLU-T increased up to a factor of 2.5 (all p<0.01) compared to controls. In treated females the activity of the monooxygenase EROD and ALD were significantly and that of the ECOD was slightly reduced. The activity of the EH was 3.7 fold, that of GS-T 2.6 fold and that of UDP-GLU-T 1.9 fold increased. The induction pattern of 10000 ppm female rats was different to that in female mice (1997i), where the cytochrome P450-dependent liver enzymes were significantly induced, indicating a possible species specific difference. The liver protoporphyrin IX content was not increased in males and females, indicating that the porphyrin metabolism was not changed by the treatment in rats. At termination ChE activity in the brain was significantly inhibited (70%) in females and slightly inhibited in males (20%). The plasma ChE activity was not markedly inhibited in males (8%), but in females it was lower (63%) compared to controls. In erythrocytes ChE activity was significantly inhibited in both males (82%) and females (94%).

In conclusion the test substance was a strong inducer of Phase II enzymes and a ChE inhibitor when given at high doses to rats.

- rats, effects on bile flow and composition and liver function

A sub-acute special mechanistic GLP-conform toxicity study in rats was performed to investigate effects of the test substance on bile flow, bile composition and liver function parameters in bile duct cannulated rats (2000c). Reasons for the conduction were findings in previous dog studies, in which the test substance had caused effects on the gall bladder mucosa, which were ascribed to changes in hepatic bile acid synthesis. An induced activity of hepatic cholesterol 7alpha- hydroxylase (CYP7A), the rate limiting enzyme in the conversion of cholesterol to bile acids, was also identified in dogs. However, previous rat studies revealed no suspicious findings on biliary physiology. Therefore, focus of this study was measuring hepatic CYP7A activity and bile composition in rats with the aim to compare the situation in both species.

In this study the test substance was administered in the diet to groups of 15 male Wistar rats at target concentrations of 6400 ppm (equal to 580.9 mg/kg bw/day) for up to 7 weeks. General examinations included daily clinical observations and weekly determination of feed consumption and body weight. Bile flow was determined and bile samples were collected via bile duct cannulation for 4 h at the end of study. Gross and histopathology of liver tissue were performed on all animals. In addition liver tissue was used for determination of microsomal CYP7A activity. In the bile samples, contents of the test substance, its metabolites and bile acids were determined.

Clinical findings or effects on survival rates were not observed in treated rats. At a dose of 6400 ppm feed intake of treated rats was increased and body weight development was retarded (approx. 10%), indicating that this is the maximum tolerated dose. Rats treated at a dose of 6400 ppm showed increased liver weights and histological changes (more homogeneous/fine granular cytoplasm of centrilobular hepatocytes) which correspond to the microsomal (Phase II) liver enzyme induction determined in previous rat studies. In addition, a minimal fat accumulation in periportal hepatocytes was also observed in several treated rats. An increased absolute bile flow occurred in treated rats, which corresponded to the increased liver weight, was considered a secondary effect. No primary effect of the test substance on bile flow was determined.

In the bile fluid, no test substance and only low concentrations of the metabolites o-chlorophenyltetrazolinone (CPT) and cyclohexyl-o-glucuronide were detected.

No induction of CYP7A activity and no significantly increased bile acid content was determined in treated rats, which was in line with the results of previous conducted rat studies. The results in rats were in contrast to the results of mechanistic studies in beagle dogs. In these studies an increase of hepatic CYP7A activity, of bile flow, and of several unconjugated bile acids were observed and these effects were causally related to changes in gall bladder mucosa.

This species difference can be explained by the different pattern of hepatic microsomal enzyme induction: in dogs, mainly the induction of Phase I enzymes were observed, whereas in rats the induction of Phase II enzymes was more prominent.

Expert statements

- evaluation of oncogenic effects in urinary bladder in rats

Based on carcinogenicity studies, chronic toxicity studies and mechanistic studies in mice and rat the following was summarized and concluded in an expert statement on carcinogenicity (1999b). In mice no carcinogenic potential of the test substance was observed in a 2-year carcinogenicity study (1997n).

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 cyclohexylamine (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.

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

- evaluation of gallbladder effects in mice and dogs

In an expert statement gallbladder effects were summarized and evaluated also taken into account genotoxicity studies with the test substance and its metabolites (2000e). Hyperplasia of the gallbladder epithelium was observed in higher doses of sub-chronic and chronic studies in mice and dogs, but not in the rat. Several special studies were conducted to elucidate the mechanism of gallbladder epithelium hyperplasia and are summarized in the following.

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.

Conclusion

In conclusion, the dog can be regarded as the most sensitive species with the chronic NOAEL being 20 ppm (corresponding to 0.52 mg/kg bw/day in females) with the liver being the target organ in all three species.

Thus, the target substance is considered to meet the classification criteria for STOT RE 2, H373, liver according to Regulation (EC) No. 1272/2008 (CLP).

Repeated dose toxicity: dermal

- sub-acute study, rat, 28 d

A sub-acute dermal toxicity study in compliance with GLP and according to OECD 410 was performed in male and female Wistar rats (1997k). In this study 5 rats per dose group and sex were administered the test substance under semi-occlusive conditions without any vehicle, but moistened with water at doses of 50, 250 and 1000 mg/kg bw/day. Animals were treated for a period of 4 weeks and animals of an additional recovery group (controls and rats treated at 1000 mg/kg bw/day) were left untreated for 2 further weeks. In the first 3 weeks of the study rats were treated 5 days/week and in the last week 7 days/ week for 6 h/day, respectively.

Examinations included clinical observations, mortalities, dermal irritation, body weight, food consumption, haematology and clinical biochemistry analysis, gross pathology and histopathology examinations including determination of organ weights.

No effect on clinical signs, dermal irritation, mortality and body weight were observed. An increased food uptake was observed in males of the recovery group dosed at 1000 mg/kg bw/day. Since changes in food uptake were not visible in the main groups the increased uptake was not considered treatment-related.

Haematology analysis revealed statistically significantly decreased mean corpuscular volume in females of dosed at 250 and 1000 mg/kg bw/day, statistically decreased haematocrit in females of the 250 mg/kg bw/day group, statistically increased mean corpuscular haemoglobin concentration in females of the 50 and 250 mg/kg bw/day and statistically decreased platelet counts in females of the 50 mg/kg bw/day dose group. All haematological findings were only observed in one sex and did not show dose-dependency and were therefore not considered treatment-related.

At a dose of 1000 mg/kg bw/day significant inhibition of cholinesterase activity by more than 20% in erythrocytes was present in males and females. In brain tissue statistically significant inhibition (10%) was observed at 1000 mg/kg bw/day in females only. Analysis of the recovery group revealed that these effects were reversible within the 2-week recovery period.

Based on these findings a local NOAEL of 1000 mg/kg bw/day and a systemic NOAEL of 250 mg/kg bw/day were determined.

- sub-acute study, rat, 12 d

A dose-range finding study for a sub-acute, dermal toxicity was performed in female rats (1996d). In this study rats were administered the test substance at doses of 100, 300 and 1000 mg/kg bw/day for a period of 12 days. In the first week animals were treated 5 days per week and in the second week 7 days per week for 6 h per days, respectively.

Examinations included clinical findings, dermal irritation parameters, body weight, feed intake, clinical biochemistry parameters, organ weights and macroscopical findings. At all dose levels inhibition of cholinesterase activity in the plasma, erythrocytes and brain was observed and the effects were dose-dependent (inhibition plasma: 23% (100 mg/kg bw/day), 30% (300 mg/kg bw/day), 33% (1000 mg/kg bw/day), inhibition erythrocytes: 21% (100 mg/kg bw/day), 33% (300 mg/kg bw/day), 42% (1000 mg/kg bw/day) and inhibition brain: 2% (100 mg/kg bw/day), 2% (300 mg/kg bw/day), 9% (1000 mg/kg bw/day). In addition an increase of adrenal gland weights was observed at a dose of 1000 mg/kg bw/day. Further effects were not observed. Based on these findings a dose range of 50, 250 and 1000 mg/kg bw/day was considered appropriate for the sub-acute dermal toxicity study. Due to insufficient documentation (only abstract available) the study was not considered for hazard evaluation.

Conclusion

Based on the findings in the sub-acute dermal toxicity study in rats a local dermal NOAEL of 1000 mg/kg bw/day (corresponding to 7.5 mg/cm2) and a systemic dermal NOAEL of 250 mg/kg bw/day were determined.

References:

(ECHA, 2016) ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance Draft Version 6.0 May 2016

Justification for classification or non-classification

Based on the available data, the target substance is considered to meet the classification criteria for STOT RE 2, H373, liver according to Regulation (EC) No. 1272/2008 (CLP).

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)	control	20	40	200	750
Mean absolute organ weights [g] - males
body weight (terminal)	13179.5	13592.0	13734.3	13973.7	12057.8
adrenals	1.215	1.083	1.308	1.354	1.495
brain	81.850	79.825	81.325	82.733	78.200
heart	93.950	90.650	98.325	99.100	86.125
kidney	59.100	60.350	63.225	56.433	63.600
liver	355.400	390.100	41.7025	470.633*	611.600*
lungs	89.550	113.500	104.200	101.033	93.350
spleen	84.900	66.450	80.450	95.733	74.350
testes	17.896	14.777	18.603	16.762	17.712
pituitary	0.070	0.069	0.065	0.067	0.064
thyroid	0.911	1.105	1.033	1.401	1.566
thymus	7.914	10.734	11.755	11.764	10.028
Mean absolute organ weights [g] - females
body weight (terminal)	13100.8	12083.3	11768.0	13296.5	10259.5
adrenals	1.460	1.240	1.400	1.339	1.674
brain	76.575	73.875	78.625	76.600	76.600
heart	80.775	79.550	85.075	76.575	75.950
kidney	51.525	50.075	49.750	54.150	46.425
liver	342.150	309.225	388.525	384.050	457.400
lungs	87.000	82.950	92.400	82.975	80.650
spleen	92.350	90.200	95.325	71.125	51.250
ovaries	1.119	0.895	1.225	0.980	0.868
pituitary	0.065	0.060	0.068	0.071	0.062
thyroid	0.782	0.975	0.915	1.272*	1.155*
thymus	9.200	11.242	9.226	12.085	7.110
Mean relative organ weights [g] - males
adrenals	0.009	0.008	0.010	0.010	0.012*
brain	0.624	0.601	0.598	0.593	0.664
heart	0.729	0.689	0.716	0.707	0.723
kidney	0.457	0.451	0.459	0.406	0.540
liver	2.738	2.937	3.025	3.358	5.059*
lungs	0.690	0.840	0.753	0.725	0.788
spleen	0.646	0.509	0.582	0.681	0.641
testes	0.138	0.113	0.135	0.120	0.149
pituitary	0.0005	0.0005	0.0005	0.0005	0.0005
thyroid	0.0070	0.0083	0.0075	0.0101	0.0129*
thymus	0.058	0.076	0.084	0.086	0086
Mean relative organ weights [g] - females
adrenals	0.011	0.011	0.012	0.010	0.017
brain	0.612	0.625	0.677	0.576	0.751
heart	0.626	0.659	0.735	0.577	0.756
kidney	0.400	0.424	0.423	0.408	0.454
liver	2.609	2.608	3.284*	2.890	4.439*
lungs	0.686	0.683	0.796	0.626	0.800
spleen	0.721	0.723	0.829	0.536	0.519
ovaries	0.009	0.007	0.011	0.007	0.008
pituitary	0.0005	0.0005	0.0006	0.0005	0.0006
thyroid	0.060	0.0082*	0.0078	0.0097*	0.0112*
thymus	0.072	0.096	0.079	0.089	0.069

Dose (ppm)		control		20		40		200		750
sex		m	f	m	f	m	f	m	f	m	f
number		4	4	4	4	4	4	4	4	4	4
organ	Incidence [%]
Adrenals	within normal limits	4 (100)	4 (100)	4 (100)	4 (100)	3 (75)	4 (100)	4 (100)	4 (100)	4 (100)	4 (100)
	cyst	0 (0)	0 (0)	0 (0)	0 (0)	1 (25)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)
Brain	within normal limits	4 (100)	4 (100)	4 (100)	4 (100)	4 (100)	4 (100)	2 (50)	4 (100)	4 (100)	4 (100)
	dilated	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	2 (50)	0 (0)	0 (0)	0 (0)
Gall blader	within normal limits	4 (100)	4 (100)	4 (100)	4 (100)	4 (100)	4 (100)	4 (100)	4 (100)	3 (75)	4 (100)
	abnormal consistency	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (25)	0 (0)
Liver	within normal limits	4 (100)	4 (100)	4 (100)	4 (100)	4 (100)	4 (100)	3 (75)	4 (100)	3 (75)	3 (75)
	discoloration	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (25)	0 (0)	1 (25)	0 (0)
	enlarged	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (25)
Lungs	within normal limits	4 (100)	4 (100)	2 (50)	4 (100)	2 (50)	4 (100)	4 (100)	3 (75)	4 (100)	4 (100)
	discoloration	0 (0)	0 (0)	2 (50)	0 (0)	1 (25)	0 (0)	0 (0)	1(25)	0 (0)	0 (0)
	discolored zone	0 (0)	0 (0)	0 (0)	0 (0)	1 (25)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)
lymph node, mesenteric	within normal limits	4 (100)	4 (100)	4 (100)	3 (75)	4 (100)	4 (100)	4 (100)	4 (100)	4 (100)	4 (100)
	discoloration	0 (0)	0 (0)	0 (0)	1 (25)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)
Pituitary	within normal limits	4 (100)	4 (100)	3 (75)	4 (100)	4 (100)	4 (100)	4 (100)	3 (75)	4 (100)	3 (75)
	cyst	0 (0)	0 (0)	1 (25)	0 (0)	0 (0)	0 (0)	0 (0)	1 (25)	0 (0)	1 (25)
Small intestine	within normal limits	4 (100)	4 (100)	4 (100)	4 (100)	4 (100)	4 (100)	4 (100)	4 (100)	4 (100)	3 (75)
	mass	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (25)
Spleen	within normal limits	3 (75)	4 (100)	4 (100)	4 (100)	4 (100)	4 (100)	3 (75)	3 (75)	4 (100)	4 (100)
	raised zone	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (25)	0 (0)	0 (0)	0 (0)
	discolored zone	1 (25)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (25)	0 (0)	0 (0)
Stomach	within normal limits	4 (100)	4 (100)	4 (100)	4 (100)	4 (100)	4 (100)	4 (100)	4 (100)	4 (100)	4 (100)
	stenosed	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (25)	0 (0)	0 (0)	0 (0)
Thymus	within normal limits	3 (75)	4 (100)	4 (100)	4 (100)	4 (100)	4 (100)	3 (75)	4 (100)	4 (100)	3 (75)
	reduced in size	1 (25)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (25)	0 (0)	0 (0)	1 (25)
external observations	emaciated	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (100)	0 (0)	0 (0)	0 (0)

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 (ppm)		control		20		40		200		750
sex		m	f	m	f	m	f	m	f	m	f
number tissues examined		4	4	4	4	4	4	4	4	4	4
organ	lesion
Adrenals	no abnormality	4	1	3	3	2	2	4	3	1	2
	cyst	-	-	-	-	1(3)	-	-	-	-	-
	vacuolization,cytoplasm	-	3(1)	1(1)	1(1)	1(1)	2(1)	-	1(1)	3(2.7)	2(2)
Bone femur	no abnormality	4	4	4	4	4	4	4	4	4	3
	osteosclerosis	-	-	-	-	-	-	-	-	-	1(1)
Bone, sternum	no abnormality	4	4	4	4	4	3	4	4	4	4
	chondrodegeneration	-	-	-	-	-	1(1)	-	-	-	-
Brain	no abnormality	3	4	4	4	4	3	2	4	4	4
	dilatation	-	-	-	-	-	-	2(2.5)	-	-	-
	gliosis	1(1)	-	-	-	-	1(1)	-	-	-	-
Epididymides	no abnormality	4	-	4	-	4	-	3	-	4	-
	aspermia	-	-	-	-	-	-	1(5)	-	-	-
	inflammation, lymphocytic			2(1)
Eyes	no abnormality	4	4	4	4	3	4	4	4	4	4
	synechia	-	-	-	-	1(1)	-	-	-	-	-
Gall blader	no abnormality	3	2	-	1	3	1	1	2	1	1
	cyst	-	-	-	-	-	1(1)	1(1)	-	1(1)	1(1)
	hyperplasia	-	-	-	-	-	1(1)	1(1)	-	1(1.5)	1(2)
	hyperplasia, lymphoid	-	-	-	-	1(1)	-	1 (2)	-	-	-
	infiltrate, lymphocytic	1(1)	2(1)	4(1)	3(1.3)	1(1)	3(1)	2 (1)	2(1)	2(1)	2(1)
Kidneys	no abnormality	2	3	2	2	3	4	3	2	3	3
	lipidosis, glomerular	-	-	-	-	-	-	-	-	1(3)	-
	microlith	2(1)	1(1)	2(1)	2(1)	1(1)	-	1(1)	2(1)	-	1(1)
Larynx	no abnormality	4	3	3	4	3	4	4	4	4	4
	infiltrate, lymphocytic	-	1(2)	-	-	1(2)	-	-	-	-	-
	infiltrate, chronic	-	-	1(1)	-	-	-	-	-	-	-
Liver	no abnormality	4	4	2	3	4	4	-	-	-	-
	degeneration, hydropic	-	-	-	-	-	-	-	-	1(4)	-
	degeneration, vacuolar	-	-	-	-	-	-	-	-	-	1(1)
	hepatocytomegaly	-	-	-	-	-	-	4(1)*	4(1.5)*	2(3.3)*	4(2.5)*
	inflammation, chronic	-	-	1(1)	-	-	-	-	-	-	2(1)
	microgranuloma	-	-	1(1)	1(2)	-	-	-	2(1.5)	-	-
Lungs	no abnormality	3	2	3	3	2	4	4	4	4	2
	fibrosis	-	-	-	-	1(3)	-	-	-	-	-
	hyperplasia	1(2)	-	-	-	1(2)	-	-	-	-	-
	hyperplasia, adenomatous	-	-	-	1(2)	-	-	-	-	-	-

Dose (ppm)	control		20		40		200		750
sex	m	f	m	f	m	f	m	f	m	f
n	4	4	4	4	4	4	4	4	4	4
N-Demeth	0.61	1.02	1.88*	1.99*	1.65*	3.56*	3.43*	4.92*	4.99*	6.51*
O-Demeth	0.17	0.22	0.33*	0.39*	0.36*	0.59*	0.67*	0.81*	0.79*	0.82*
Cytop450	0.69	0.29	0.64	0.50	0.79	0.74*	1.51*	1.62*	1.29*	1.66*

dose (mg/kg)	day 0	day 7	day 14	day 21	day 28	sex
0	260	279	299	311	323	male
50	253	265	283	301	316	male
250	264	284	303	319	335	male
1000	253	278	295	311	321	male
0	227	227	232	234	237	female
50	225	224	230	233	236	female
250	227	224	229	233	232	female
1000	228	226	230	231	235	female

Mean feed intake – main groups
	day 7	day 14	day 21	day 28	day 7	day 14	day 21	day 28
dose (mg/kg)	g/animal/day				g/kg bw/day				sex
0	23	25	24	23	83	84	77	70	male
50	22	24*	24	24	84	84*	80	77	male
250	24	26	26	23	83	86	80	70	male
1000	23	25	25	26	83	83	80	81+	male
0	17	19	18	19	73	82	77	79	female
50	17	19	17	18	74	81	75	77	female
250	18	19	19	19	78	85	80	83	female
1000	17	19	18	19	76	84	77	83	female

Mean feed intake – recovery groups
	day 7	day 14	day 21	day 28	day 35	day 42	day 7	day 14	day 21	day 28	day 35	day 42
dose (mg/kg)	g/animal/day						g/kg bw/day						sex
0	22	23	23	23	24	23	82	82	79	76	75	73	male
1000	24	25+	25	26	25	23	87+	86+	82	81	74	68	male
0	18	19	19	21	20	17	79	83	82	86	81	72	female
1000	18	20	19	21	20	17	81	85	82	85	82	73	female

dose (mg/kg)	leuco (10⁹/L)	ery (10¹²/L)	HB (g/L)	HCT (L/L)	MCV (fl)	MCH (pg)	MCHC (g/L ery)	thro (10⁹/L)	hquick sec	sex
0	7.6	8.86	148	0.473	53.3	16.7	314	894	31.8	male (main group)
50	7.3	8.74	150	0.478	54.7	17.2	314	1029	30.6	male (main group)
250	8.8	8.94	151	0.482	54.2	17.0	314	1036	31.3	male (main group)
1000	7.3	8.78	149	0.473	53.8	17.0	316	1070	32.3	male (main group)
0	6.0	8.57	146	0.490	57.1	17.0	298	1015	27.2	female (main group)
50	5.8	8.51	145	0.466	54.8	17.0	310++	842+	27.1	female (main group)
250	5.9	8.54	139	0.454+	53.2++	16.3	306+	892	26.8	female (main group)
1000	5.4	8.44	138	0.455	53.9+	16.3	303	952	26.4	female (main group)
0	8.3	9.14	148	0.481	52.6	16.2	308	1100	26.2	male (recovery group)
1000	7.1	8.90	146	0.479	53.8	16.4	305	920	26.1	male (recovery group)
0	4.4	8.18	139	0.448	54.7	17.0	311	901	24.9	female (recovery group)
1000	5.5	8.35	140	0.446	53.4	16.7	314	910	25.2	female (recovery group)

dose (mg/kg)	CHE plasma kU/L	CHE plasma %	CHE erythrocytes kU/L	CHE erythrocytes %	CHE brain kU/L	CHE brain %	sex
0	0.41	-	0.83	-	12.00	-	male
50	0.41	0	0.74	-11	12.64+	+5	male
250	0.43	+5	0.73	-12	12.36	+3	male
1000	0.46	+12	0.62+	-25+	1165	-3	male
0	1.95	-	0.63	-	12.09	-	female
50	1.75	-10	0.65	+3	11.75	-3	female
250	1.92	-2	0.62	-2	11.42	-6	female
1000	1.55	-21	0.41++	-35++	10.88+	-10	female
0	0.55	-	1.39	-	12.15	-	male (recovery group)
1000	0.50	-10	1.02	-27	11.92	-2	male (recovery group)
0	1.98	-	1.04	-	11.49	-	female (recovery group)
1000	2.13	+8	1.10	+6	11.17	-3	female (recovery group)

Absolute organ weights (mg)
dose (mg/kg)	body weight (g)	brain	adrenals	heart	lung	liver	spleen	thymus	kidneys	testes	sex
0	323	1843	43	1062	1665	12956	602	367	2136	3062	male (main group)
50	316	1699	43	1042	1542	12481	637	401	1920	3075	male (main group)
250	335	1706	42	1035	1730	12907	661	437	2069	3208	male (main group)
1000	321	1769	47	1069	1713	13668	633	487	2191	3468+	male (main group)
0	237	1672	64	870	1399	9160	521	309	1543	-	female (main group)
50	236	1727	64	848	1293	8685	504	304	1558	-	female (main group)
250	232	1779	67	832	1225	9312	449	253	1494	-	female (main group)
1000	235	1753	59	865	1268	9245	500	305	1526	-	female (main group)
0	320	1788	37	972	1613	11918	608	407	2092	3346	male (recovery group)
1000	342	1833	41	1107	1586	13340	623	407	2291	3315	male (recovery group)
0	242	1738	59	941	1275	8791	472	334	1608	-	female (recovery group)
1000	237	1639	57	877	1264	8543	546	356	1451	-	female (recovery group)
Relative organ weights (mg)
dose (mg/kg)	body weight (g)	brain	adrenals	heart	lung	liver	spleen	thymus	kidneys	testes	sex
0	323	579	13	330	519	4013	188	112	663	960	male (main group)
50	316	538	14	329	486	3944	202	127	607	972	male (main group)
250	335	512	13	310	517	3858	199	131	621	961	male (main group)
1000	321	556	14	332	531	4250	197	152+	683	1085	male (main group)
0	237	708	27	368	591	3871	220	131	653	-	female (main group)
50	236	731	27	359	547	3676	213	129	660	-	female (main group)
250	232	767	29	358	527	4005	193	109	644	-	female (main group)
1000	235	745	25	367	539	3933	213	130	649	-	female (main group)
0	320	560	12	303	503	3726	189	126	655	1046	male (recovery group)
1000	242	536	12	323	465	3894	183	229	670	971	male (recovery group)
0	242	720	24	389	528	3636	195	138	666	-	female (recovery group)
1000	237	693	24	369	533	3594	229	150	612	-	female (recovery group)

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

Endpoint summary

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

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Reference 1

Reference 2

Endpoint conclusion

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint conclusion

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint conclusion

Additional information

Justification for classification or non-classification

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