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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

Referenceopen allclose all

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

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

Dose (ppm)

0

50

200

1000

3000

sex (male)

Weeks 0-52 (n = 60)

Week 26

0

1

0

0

0

Week 53

0

1

0

2

1

Weeks 53-104 (n = 50)

Week 79

5

4

1

5

1

Week 104

19

17

21

17

13

Dose (ppm)

0

50

200

1000

4000

sex (female)

Weeks 0-52 (n = 60)

Week 26

0

0

0

0

0

Week 53

0

2

1

0

2

Weeks 53-104 (n = 50)

Week 79

3

7

8

8

9

Week 104

13

19

22

20

21

 

Table 2: Body weights [g]

sex

m

m

m

m

m

f

f

f

f

f

Dose (ppm)

0

50

200

1000

3000

0

50

200

1000

4000

0

1

2

3

4

5

6

7

8

9

10

11

12

13

15

17

19

21

23

25

27

29

31

33

35

37

39

41

43

45

47

49

51

53

55

57

59

61

63

65

67

69

71

73

75

77

79

81

83

85

87

89

91

93

95

97

99

101

103

105

155

203

239

272

299

322

336

362

378

382

393

403

412

427

435

456

463

471

472

477

482

492

492

502

500

510

515

519

509

517

519

523

526

531

532

535

535

542

542

542

543

553

552

553

558

550

551

558

555

559

554

553

550

549

550

553

551

544

542

543

156

203

243

276

304

325

344

362

378

394

406

417

428

436

448

465

468

479

480

486

494

498

503

509

507

515

518

521

519

523

521

528

532

530

531

534

534

536

540

539

541

548

549

548

550

552

543

551

549

548

542

537

529

530

529

534

530

520

508 +

511

155

203

245

278

306

329

348

364

378

395

406

417

423

430

444

459

463

470

480

487

493

495

503

507

509

508

514

516

515

518

520

524

529

533

534

538

539

539

541

541

542

547

545

541

544

539

536

536

538

532

520 +

522

513 +

527

528

522

519

510 +

514

529

154

200

241

275

301

326

341

356

368

385

401

400

417

420

438

448

455

464

472

480

483

488

496

495

502

509

507

510

512

512

513

519

522

525

527

530

534

533

532

538

542

549

547

547

550

550

552

556

555

553

549

547

533

542

535

531

528

523

518

515

152

189++

238

264

289

312

330

344++

360++

372

382

391

401

401++

421

427++

441+

449+

454

463

466

468+

480

479+

485

483++

486++

489++

492

490++

490++

496++

500++

502++

505+

506++

513

511++

512++

515+

516+

521++

519++

516++

518++

512 ++

523 +

524 ++

527 +

526 +

518 +

513 ++

493 ++

504 ++

498 ++

497 ++

492 ++

495 ++

491 ++

483 ++

130

149

163

176

186

196

205

212

217

222

225

229

234

242

242

247

249

255

258

260

261

267

271

277

276

280

284

286

289

290

295

296

301

302

304

309

311

312

314

315

319

323

322

325

329

333

328

333

333

336

327

327

329

327

326

328

323

323

318

313

127

145

161

173

183

191

200

208

212

216

222

226

230

234

240

243

247

251

254

257

257

261

266

271

273

275

279

281

284

285

289

288

291

291

294

298

300

303

307

307

309

316

314

318

321

323

321

329

330

331

326

325

324

324

325

327

325

324

315

325

130

150

166

179

190

199

207

211

217

223

228

232

238

239

244

249

252

258

260

263

264

266

272

276

280

281

285

288

289

289

293

293

297

302

304

307

309

308

312

315

316

322

322

325

327

328

327

332

333

337

332

330

331

327

329

326

325

320

312

341

128

147

164

178

186

196

203

207

213

219

221

228

231

234

237

242

246

249

252

254

257

257

261

266

270

270

274

276

276+

275+

280+

279++

282++

284+

287+

291+

292+

294+

300

301

303

308

307

311

313

314

310

320

320

320

316

314

317

317

313

311

311

312

307

320

130

147

167

179

189

196

202

205

209

215

217

222

225

226++

230+

234++

234++

238++

242++

245++

246++

248++

252++

253++

257++

258++

262++

264++

264++

263++

266++

266++

268++

270++

271++

274++

275++

279++

278++

280++

281++

285++

283++

283++

286++

284 ++

284 ++

289 ++

291 ++

288 ++

286 ++

288 ++

287 ++

289 ++

289 ++

286 ++

283 ++

284 ++

281 ++

295

+ difference against control for p<0.05

++ difference against control for p<0.01

 

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

Intake food

 

 

 

g/animal

g/kg bw

Dose (ppm)

Sex

Days

Total

per Day

Total

per Day

0

m

708

14715

20.8

35813

50.6

50

m

708

14923

21.1

35653

50.4

200

m

708

15169

21.4

36331

51.3

1000

m

708

15391

21.7

37307

52.7

3000

m

708

15807

22.3

40218

56.8

0

f

708

12072

17.1

48515

68.5

50

f

708

12522

17.7

51527

72.8

200

f

708

12729

18.0

51524

72.8

1000

f

708

12712

18.0

53372

75.4

4000

f

708

13177

18.6

57821

81.7

Intake of test compound

 

 

 

mg/animal

mg/kg bw

Dose (ppm)

Sex

Days

Total

per Day

Total

per Day

50

m

708

746

1.1

1783

2.5

200

m

708

3034

4.3

7266

10.3

1000

m

708

15391

21.7

37307

52.7

3000

m

708

47421

67.0

120654

170.4

50

f

708

626

0.9

2576

3.6

200

f

708

2546

3.6

10305

14.6

1000

f

708

12712

18.0

53372

75.4

4000

f

708

52707

74.4

231285

326.7

 

Table 4: Mean daily cumulative water intake

Intake of water

 

 

 

g/animal

g/kg bw

Dose (ppm)

Sex

Days

Total

per Day

Total

per Day

0

m

708

18267

25.9

40827

57.8

50

m

708

18971

26.9

41396

58.6

200

m

708

19254

27.3

42187

59.8

1000

m

708

19034

27.0

42158

59.7

3000

m

708

19391

27.5

44915

63.6

0

f

708

18478

26.2

67633

96.8

50

f

708

18745

26.6

70078

99.3

200

f

708

18848

26.7

68707

97.3

1000

f

708

18539

26.3

70478

99.8

4000

f

708

17344

24.6

69761

98.8

 

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

sex

m

m

m

m

f

f

Dose (ppm)

0

200

1000

3000

0

4000

No. of eyes examined

61

71

61

73

76

58

Turbid cornea, including vascularisation of cornea

1.6

7.0

1.6

8.2

0

3.4

Turbid cornea, including vascularisation of cornea

Snow ball like turbidities between lens and vitreous

body (= posterior lenticular rupture)

11.4

4.2

8.2

13.7

7.9

0

Diffuse retrolenticular opacity (all degrees)

24.6

9.8

18.0

17.8

10.5

8.6

Wedge shaped waterclefts in the cortex, slight

18.0

7.0

8.2

8.2

3.9

5.1

Wedge shaped waterclefts in the cortex, moderate

0

0

3.3

1.4

0

1.7

Lens opacity in the whole cortex, slight

1.6

1.4

0

2.7

2.6

1.7

Lens opacity in the whole cortex, moderate

1.6

1.4

1.6

5.5

3.9

0

Lens opacity in the whole cortex, severe

1.6

0

6.5

5.5

6.6

3.4

 

Table 6: Hematology

Dose

 

 

LEU

ERY

HB

HCT

MCV

MCH

MCHC

RETI

HEINZ

THRO

HQUICK

ppm

Sex

Week

109/L

109/L

g/L

L/L

fl

pg

g/LERY

0/00

0/00

109/L

sec

0

m

26/27

10.3

9.04

147

0.459

50.8

16.3

320

18

0

1040

25.4

50

m

26/27

8.7

8.91

147

0.453

50.9

16.5

324

19

0

998

25.2

200

m

26/27

8.3

8.51

141

0.439

51.6

16.5

320

18

0

1109

24.7

1000

m

26/27

8.3

9.06

141

0.434

48.0++

15.6

325

17

0

1053

26.2

3000

m

26/27

8.0

8.58

138+

0.425++

49.6

16.1

325

17

1

982

25.4

0

m

52

9.5

9.42

153

0.487

51.8

16.2

313

16

0

1082

26.3

50

m

52

8.5

9.17

149

0.479

52.3

16.3

312

17

0

1059

26.0

200

m

52

8.4

8.87+

148

0.471

53.1

16.7

314

18

0

1172

26.1

1000

m

52

7.4

9.37

146

0.462

49.4

15.6

315

20

0

1082

25.4

3000

m

52

8.2

9.29

146

0.472

50.8

15.7

309

20

0

1026

26.3

0

m

78

8.0

9.04

151

0.476

52.7

16.8

318

17

0

1091

24.7

50

m

78

7.3

8.99

149

0.472

52.5

16.6

316

14

0

1073

24.6

200

m

78

6.7

8.20+

141

0.441

53.7

17.2

319

16

0

1277

24.0

1000

m

78

6.4

8.93

141

0.441

49.5+

15.9+

321

17

0

1041

24.1

3000

m

78

7.5

8.77

139

0.440

50.2

15.9+

316

15

0

998

24.1

0

m

103/104

9.2

9.26

160

0.512

55.4

17.3

312

20

0

1220

25.9

50

m

103/104

7.9

8.63

148

0.457+

52.9

17.1

322++

27

0

1158

25.0

200

m

103/104

7.9

8.85

154

0.473

53.5

17.5

326++

16

0

1156

24.2

1000

m

103/104

7.3

8.73

144

0.443++

50.9++

16.5

324++

23

0

1186

24.8

3000

m

103/104

6.9

9.07

150

0.466

51.5++

16.5

321+

29

0

1081

25.8

0

f

26/27

6.0

8.21

143

0.439

53.5

17.4

325

15

0

915

24.5

50

f

26/27

6.2

7.94

136

0.416

52.4

17.1

328

15

0

978

23.7

200

f

26/27

5.9

8.10

139

0.422

52.2

17.2

330

14

0

979

23.8

1000

f

26/27

6.2

8.01

137

0.424

52.8

17.1

325

17

0

955

23.8

4000

f

26/27

5.8

8.57

139

0.434

50.6++

16.2++

320

17

0

1046++

22.4

 

0

f

52

4.6

8.03

140

0.440

54.8

17.4

318

20

0

809

24.7

50

f

52

5.8

8.15

138

0.437

53.6

17.0

317

18

0

921

25.4

200

f

52

5.7

8.26

142

0.440

53.4

17.2

322

17

0

956

25.2

1000

f

52

5.7

8.25

140

0.447

54.2

17.0

314

19

0

897

24.1

4000

f

52

5.8

8.36

136

0.440

53.1

16.4+

309++

20

0

1017+

23.8

0

f

78

4.1

8.33

146

0.454

54.6

17.5

321

25

0

858

23.2

50

f

78

4.6

7.79

136

0.429

56.6

17.8

316

44

0

795

23.5

200

f

78

5.6

8.24

142

0.443

53.8

17.3

321

31

0

840

23.4

1000

f

78

4.3

8.16

143

0.451

55.4

17.6

317

31

0

848

26.2++

4000

f

78

4.7

8.06

133

0.431

53.6

16.6

309++

36

0

930

24.8+

0

f

103/104

5.6

8.29

151

0.453

54.9

18.3

333

31

0

1003

24.0

50

f

103/104

4.7

8.34

151

0.454

54.5

18.1

333

28

0

984

23.7

200

f

103/104

6.8

8.22

149

0.450

54.7

18.1

330

26

0

994

24.6

1000

f

103/104

4.3

8.33

151

0.463

55.7

18.1

325

29

0

948

24.8

4000

f

103/104

5.1

8.22

143  

0.443

 54.2 

17.4 

322+ 

 31 

0 

1066 

23.9 

+ difference against control for p<0.05

++ difference against control for p<0.01

 

Table 7: Differential blood count

Dose

Week

Sex

LYM

SEGM

EOS

MONO

BAND

ppm

 

 

%

%

%

%

%

0

26/27

m

87.5

8.0

0.2

4.3

0.0

50

26/27

m

89.8

6.5

0.3

3.4

0.0

200

26/27

m

86.0

10.1

0.6

3.3

0.0

1000

26/27

m

87.9

8.1

0.7

3.3

0.0

3000

26/27

m

89.4

7.6

0.6

2.5

0.0

0

52

m

79.7

12.7

1.0

6.6

0.0

50

52

m

78.8

11.7

1.8

7.7

0.0

200

52

m

79.1

12.7

0.9

7.3

0.0

1000

52

m

77.5

13.6

1.4

7.6

0.0

3000

52

m

78.0

13.7

0.6

7.8

0.0

0

78

m

78.4

13.9

1.5

6.2

0.0

50

78

m

75.9

16.2

2.2

5.6

0.0

200

78

m

74.9

17.4

1.7

6.2

0.0

1000

78

m

74.7

18.9

1.1

5.3

0.0

3000

78

m

77.6

17.0

0.8

4.6

0.0

0

103/104

m

67.8

23.5

1.2

7.4

0.0

50

103/104

m

62.7

29.7

1.7

6.0

0.0

200

103/104

m

70.0

23.6

1.5

4.8

0.1

1000

103/104

m

66.6

25.4

2.6

5.5

0.0

3000

103/104

m

71.8

23.0

1.2

4.2

0.0

0

26/27

f

88.4

8.4

0.8

2.5

0.0

50

26/27

f

86.0

11.3

0.8

1.9

0.0

200

26/27

f

89.5

7.8

0.8

1.9

0.0

1000

26/27

f

90.7

8.2

0.4

0.7+

0.0

4000

26/27

f

88.7

8.5

0.8

2.0

0.0

0

52

f

79.9

14.0

1.4

4.7

0.0

50

52

f

76.5

16.5

1.0

6.0

0.0

200

52

f

80.7

13.3

1.2

4.8

0.0

1000

52

f

80.5

15.0

0.6

3.9

0.0

4000

52

f

86.4

9.7

0.5

3.4

0.0

0

78

f

71.8

23.7

1.2

3.3

0.0

50

78

f

75.7

16.2

1.7

6.4

0.1

200

78

f

65.9

26.9

1.0

6.3

0.0

1000

78

f

73.5

19.8

1.2

5.6

0.0

4000

78

f

78.4

16.0

1.1

4.5

0.0

0

103/104

f

70.4

27.4

1.2

1.1

0.0

50

103/104

f

69.3

28.8

0.8

1.1

0.0

200

103/104

f

60.4

37.3

1.3

1.0

0.0

1000

103/104

f

75.8

22.0

1.0

1.2

0.0

4000

 103/104

f

73.1

 23.8

 1.6

 1.6

0.0

+ difference against control for p<0.05

++ difference against control for p<0.01

 

Table 8: Clinical Chemistry

Dose

Week

Sex

ASAT (GOT)

ALAT (GPT)

APh

GGT

GLDH

ppm

 

 

U/L

U/L

U/L

U/L

U/L

0

26/27

m

41.3

38.9

190

1

5.7

50

26/27

m

38.8

40.1

186

1

5.4

200

26/27

m

44.4

40.3

171

2

7.9

1000

26/27

m

38.5

40.4

157+

2

5.0

3000

26/27

m

35.1

37.7

150+

3++

3.8

0

52

m

39.9

42.2

184

1

16.2

50

52

m

40.5

44.l

193

1

22.8

200

52

m

36.0

37.9

162

2

12.0

1000

52

m

31.2

37.4

154+

1

12.6

3000

52

m

34.6

41.0

156+

2

12.8

0

78

m

38.8

36.5

157

1

16.0

50

78

m

42.5

43.8

178

2

22.0

200

78

m

35.1

32.1

142

1

14.0

1000

78

m

33.2

36.0

141

1

17.9

3000

78

m

32.7

35.7

144

2

20.0

0

103/104

m

40.2

37.5

164

3

21.4

50

103/104

m

42.2

33.1

175

2

21.6

200

103/104

m

37.4

32.3

134

2

10.8

1000

103/104

m

37.4

29.8

137

3

14.8

3000

103/104

m

36.9

35.3

140

3

19.0

0

26/27

f

40.7

43.4

121

0

11.5

50

26/27

f

48.0

43.6

133

0

18.7

200

26/27

f

41.0

44.9

122

0

11.3

1000

26/27

f

32.3+

41.9

103

0

6.8

4000

26/27

f

30.9++

42.6

105

1

1.0

0

52

f

44.7

42.6

120

0

17.2

50

52

f

46.8

44.1

134

0

22.9

200

52

f

42.5

43.8

111

0

20.4

1000

52

f

55.4

55.2

99

0

63.3

4000

52

f

33.3

45.0

118

0

9.2

0

78

f

59.4

45.7

100

1

59.2

50

78

f

89.4

62.2

113

0

104.8

200

78

f

73.9

54.4

91

0

87.7

1000

78

f

40.3

36.3

81+

1

29.1

4000

78

f

41.2

37.5

90

2+

18.4

0

103/104

f

61.3

45.9

114

0

35.5

50

103/104

f

61.3

49.0

114

1

53.7

200

103/104

f

76.6

53.8

102

0

70.9

1000

103/104

f

45.7

41.2

92

0

26.4

4000

103/104

f

 43.4

 43.6 

102 

2++ 

41.5 

+ difference against control for p<0.05

++ difference against control for p<0.01

 

Table 9: Activities and inhibition of cholinesterases (CHE)

Dose

Week

Sex

CHE/Brain

CHE/Plasma

CHE/Erythrocytes

ppm

 

 

U/g

% Inhibition

kU/L

% Inhibition

kU/L

% Inhibition

0

27

m

-

 

0.50

 

1.06

 

50

27

m

-

 

0.52

 

1.06

 

200

27

m

-

 

0.52

 

0.88

17

1000

27

m

-

 

0.50

 

0.65+

39

3000

27

m

-

 

0.45

10

0.32++

70

0

52/53

m

10.64

 

0.62

 

1.14

 

50

52/53

m

10.46

2

0.68

 

1.15

 

200

52/53

m

10.56

 

0.63

 

1.01

11

1000

52/53

m

10.26

4

0.61

2

1.00

12

3000

52/53

m

9.54

10

0.56

10

0.57++

50

0

78

m

-

 

0.72

 

1.24

 

50

78

m

-

 

0.71

 

1.35

 

200

78

m

-

 

0.71

1

1.16

6

1000

78

m

-

 

0.63

13

0.84+

32

3000

78

m

 

 

0.55

24

0.55++

55

0

103/104/105

m

9.88

 

0.66

 

0.83

 

50

103/104/105

m

9.87

 

0.95

 

0.76

8

200

103/104/105

m

9.86

 

0.86

 

0.70

16

1000

103/104/105

m

9.47

4

0.65

2

0.50++

40

3000

103/104/105

m

8.50

14

0.55

17

0.15++

82

0

26/27

f

-

 

2.47

 

0.77

 

50

26/27

f

-

 

2.24

9

0.83

 

200

26/27

f

-

 

2.15

13

0.70

9

1000

26/27

f

-

 

2.57

 

0.38++

51

4000

26/27

f

-

 

2.30

7

0.08++

90

0

52/53

f

11.28

 

2.39

 

1.24

 

50

52/53

f

10.82+

4

2.21

8

1.17

6

200

52/53

f

10.80

4

2.39

 

0.96+

23

1000

52/53

f

9.41++

17

2.73

 

0.58++

53

4000

52/53

f

7.07++

37

2.49

 

0.34++

73

0

78

f

-

 

2.18

 

1.19

 

50

78

f

-

 

2.03

7

1.18

1

200

78

f

-

 

2.12

3

1.01

15

1000

78

f

-

 

2.45

 

0.45++

62

4000

78

f

-

 

2.22

 

0.13++

89

0

103/104/105

f

10.22

 

2.25

 

0.74

 

50

103/104/105

f

10.02

2

2.31

 

0.66

11

200

103/104/105

f

9.86

4

1.95

13

0.73

1

1000

103/104/105

f

8.91++

13

2.11

6

0.28++

62

 4000

103/104/105

f

6.02++

41

 2.08

 8

0.08++

89

+ difference against control for p<0.05

++ difference against control for p<0.01

 

Table 10: Determination of urine pH week 98/99

 

pH mean value

Sex

male

female

Dose (ppm)

0

7.55

7.20

50

7.57

7.47

200

7.32

7.12

1000

7.45

7.64

3000

7.34

-

4000

-

7.67

 

Table 11: Urinalyses

Dose (ppm)

Week

Sex

VOL (ml)

Density (g/L)

PROT*Vol (mg)

PROT (g/L)

0

26

m

13.9

1023

20.3

1.78

50

26

m

14.8

1021

30.1

2.88

200

26

m

12.2

1025

56.5

5.23

1000

26

m

10.8

1032

26.7

3.29

3000

26

m

15.6

1020

31.1

3.07

0

52

m

6.3

1036

33.5

5.73

50

52

m

8.2

1035

57.9

7.95

200

52

m

7.0

1039

101.2

13.71

1000

52

m

11.2

1032

61.4

9.03

3000

52

m

9.3

1034

58.2

7.84

0

79

m

7.1

1037

35.2

5.35

50

79

m

7.8

1030

42.9

5.49

200

79

m

12.3

1023

76.1

6.53

1000

79

m

7.5

1025

36.4

5.99

3000

79

m

8.4

1028

43.5

6.37

0

103

m

11.6

1024

75.9

7.70

50

103

m

8.6

1031

77.2

8.98

200

103

m

10.9

1024

84.1

8.65

1000

103

m

8.8

1030

91.4

9.67

3000

103

m

11.8

1024

75.7

6.64

0

26

f

8.5

1026

5.5

0.68

50

26

f

14.0

1016

2.8

0.25

200

26

f

12.5

1022

2.4

0.24+

1000

26

f

6.9

1028

1.8

0.31

4000

26

f

7.3

1026

1.9

0.34

0

52

f

8.4

1029

13.3

1.25

50

52

f

9.0

1021

3.2

0.37

200

52

f

10.7

1022

4.5

0.45

1000

52

f

12.5

1015+

2.5

0.32+

4000

52

f

8.4

1030

1.8

0.29+

0

79

f

5.8

1026

10.1

1.85

50

79

f

8.0

1020+

6.3

0.91

200

79

f

11.2+

1019+

10.9

1.25

1000

79

f

10.8+

1015+

6.0

0.66

4000

79

f

10.6

1018+

3.3

0.42

0

103

f

8.4

1024

46.0

4.94

50

103

f

10.0

1021

42.5

4.80

200

103

f

7.9

1024

23.6

2.85

1000

103

f

9.8

1022

19.7

1.16+

4000 

103

 f 

7.9

1029

7.5

1.14+

+ difference against control for p<0.05

++ difference against control for p<0.01

 

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

Dose

Sex

Body weight

Brain

Adrenals

Heart

Liver

Spleen

Kidneys

Testes

Ovaries

Absolute organ weights

g

mg

mg

mg

mg

mg

mg

mg

mg

0

m

517

2052

54

1402

17938

853

2910

3844

-

50

m

557

2159+

53

1534

19013

944

3220

4062

-

200

m

528

2064

54

1467

17702

881

3085

3815

-

1000

m

531

2128

52

1560

19351

801

3054

3652

-

3000

m

519

2105

56

1439

21255+

715

2998

4051

-

0

f

312

1912

84

1023

10016

535

1994

-

172

50

f

302

1890

83

1031

9690

561

2057

-

173

200

f

296

1920

83

1059

9442

476

2061

-

197

1000

f

285

1879

80

988

10299

684

1983

-

204

4000

f

261++

1827

69

974

11901++

488

1856

-

162

Relative organ weights

Body weight

Brain

Adrenals

Heart

Liver

Spleen

Kidneys

Testes

Ovaries

g

mg

mg

mg

mg

mg

mg

mg

mg

0

m

517

400

10

272

3480

166

561

749

-

50

m

557

390

10

276

3424

170

580

731

-

200

m

528

393

10

279

3347

167

585

723

-

1000

m

531

403

10

296

3649

151

579

697

-

3000

m

519

406

11

278

4094+

137

578

782

-

0

f

312

620

27

331

3235

173

642

-

55

50

f

302

628

28

342

3205

186

681

-

57

200

f

296

652

28

357

3196

195

696

-

68

1000

f

285

660

28

347

3615+

235

696

-

71

4000

f

261++

704++

27

375+

4584++

186

711+

-

63

+ difference against control for p<0.05

++ difference against control for p<0.01

 

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

Dose

Sex

Body weight

Brain

Adrenals

Heart

Liver

Spleen

Kidneys

Testes

Ovaries

Absolute organ weights

g

mg

mg

mg

mg

mg

mg

mg

mg

0

m

534

2176

64

1866

18013

1217

3491

3785

-

50

m

507

2191

68

1960

17363

1098

3429

3929

-

200

m

508

2172

58

1830

18083

1157

3611

3847

-

1000

m

513

2165

71

18368

18368

1035+

3407

4048

-

3000

m

483++

2118

78

19494

19494

937++

3369

4029

-

0

f

318

1931

84

11395

11395

721

2295

-

216

50

f

318

1957

77

11784

11784

714

2380

-

173

200

f

312

1947

82

11347

11347

738

2328

-

181

1000

f

308

1938

69

11579

11579

671

2197

-

193

4000

f

282++

1914

71

13476++

13476++

585++

2190

-

194

Relative organ weights

Body weight

Brain

Adrenals

Heart

Liver

Spleen

Kidneys

Testes

Ovaries

g

mg

mg

mg

mg

mg

mg

mg

mg

0

m

534

410

12

351

3369

229

655

712

-

50

m

507

435

14

387

3416

229

680

776

-

200

m

508

434

12

367

3608

230

721

756

-

1000

m

513

426

14

364

3608

203

669

793

-

3000

m

483++

483++

17

400+

4050++

196+

706

835++

-

0

f

318

617

27

448

3608

229

728

-

68

50

f

318

625

25

444

3758

229

754

-

54

200

f

312

633

27

440

3674

239

754

-

58

1000

f

308

638

23

437

3773

219

716

-

62

4000

f

282++

682++

25

470

4795++

208

781

-

69

+ difference against control for p<0.05

++ difference against control for p<0.01

 

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

Sex

 

m

m

m

m

 

f

f

f

f

f

Dose

(ppm)

0

50

200

1000

3000

0

50

200

1000

4000

Organ/Finding

Number Animals

10

10

10

10

10

10

10

10

10

10

Adrenal Glands

- increased vacuolation in zona fascicu.*

§4

6

4

6

10

§2

0

0

0

9

Liver

- hepatocellular hypertrophy (centilobul.)

§0

0

0

6

10

§0

0

0

3

10

Urinary Bladder

- transitional hyperplasia

#0

0

2

2

3

§0

0

0

0

3

*= increased cytoplasmic vacuolisation in zona fasciculata

# trend (p < 0.05)

§ trend (p < 0.01)

 

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

Sex

 

 

m

m

m

m

 

 

f

f

f

f

f

Dose

(ppm)

 

0

50

200

1000

3000

 

0

50

200

1000

4000

Organ/Finding

Number Animals

 

50

50

50

50

50

 

50

50

50

50

50

Liver

- eosinophilic foci

#

3

3

1

2

10

#

 

 

3

3

3

- focal degenerative changes (periportal)

§

 

 

 

2

13

 

 

 

 

 

1

- clear cell foci

 

36

34

31

35

28

 

6

9

6

9

1

- cytoplasmic changes (periportal)

§

 

 

 

2

22

 

 

 

 

 

 

- cytoplasmic changes (centrilobular)

§

 

 

1

 

14

§

1

3

3

8

38

- hepatocellular hypertrophy (centilobul.)

 

1

3

 

1

4

#

 

 

1

3

40

- peliotic foci

 

 

 

 

 

 

§

 

 

 

 

3

- single cell necrosis

 

 

 

 

 

 

§

4

7

5

5

16

Urinary Bladder

- transitional hyperplasia (focal)

§

 

 

 

 

4

 

1

 

 

 

3

- simple transitional hyperplasia (diffuse)

§

 

 

3

 

11

§

 

 

 

 

20

Skeletal Muscle

- atrophy

#

7

2

9

10

11

§

 

 

1

2

9

- fibre degeneration (thigh)

 

4

5

6

3

6

#

2

 

1

 

7

Sciatic Nerve

- degenerative myelinopathy

§

21

25

27

32

43

§

17

20

13

23

38

Thyroid Gland

- follicular hyperplasia

§

1

1

2

3

7

 

2

 

 

2

1

- mineralization of follicular colloid

#

33

28

31

35

39

§

13

13

17

17

43

Adrenal Glands

- increased vacuolation in zona fascicu.*

§

5

7

7

3

20

 

1

2

2

3

4

- degenerative changes in the cortex

 

5

3

3

3

3

 

19

14

11

8

4

Pituitary Gland

(pars distalis)

- hyperplasia (focal)

 

10

6

4

13

10

 

18

13

9

11

9

Stomach

(glandular mucosa)

- dilated glands

 

6

4

8

7

6

#

7

7

6

8

16

Tongue

- necrotizing arteritis

 

7

12

7

11

9

 

7

7

4

2

0

Heart

- cardiomyopathy

 

43

45

48

44

48

 

37

37

33

28

22

Kidneys

- chronic progressive nephropathy

 

47

47

48

44

48

 

41

32

36

26

21

- diffuse urothelial hyperplasia

 

10

18

16

8

15

 

35

35

30

21

19

Spleen

- pigment storage

 

19

24

31

20

27

§

9

9

11

15

23

# trend (p < 0.05)

§ trend (p < 0.01)

 

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

Sex

 

Males

Females

Dose

(ppm)

0

50

200

1000

3000

0

50

200

1000

4000

Organ/Findings

No.animals

50

50

50

50

50

50

50

50

50

50

 

Liver

No. exam.

50

 

49

 

50

 

50

 

50

 

50

 

49

 

50

 

50

 

50

- Hepatocellular Adenoma

 

1

 

1

 

 

 

1

 

 

1

- Hcpatocellular Adcnocarcinoma

 

1

 

 

 

 

 

 

 

 

 

-Cholangioma

 

 

 

 

 

 

 

 

 

1

 

- Cholangiocarcinoma

 

 

1

 

 

 

 

 

 

 

 

Heart

No. exam.

50

49

50

50

50

50

49

50

50

50

- Endocardial Tumor (mal.)

 

2

1

 

 

 

 

1

 

 

 

Kidneys

No. exam.

50

49

50

50

50

50

49

50

50

50

-Adenoma

 

 

 

 

1

 

 

 

 

 

 

- Lipoma

 

 

 

 

 

1

 

 

 

 

 

- Liposarcoma

 

 

 

1

 

 

 

 

 

 

 

Urinary Bladder

No. exam.

50

48

50

50

50

50

49

48

49

49

- Transitional Cell Papilloma

 

 

 

 

 

 

§

 

 

 

2

- Transitional Cell Carcinoma

 

 

 

 

 

 

 

 

 

 

1

Urethra (residual)&)

No. exam.

 

 

 

 

1

 

 

 

 

 

- Transitional Cell Carcinoma

 

 

 

 

 

1

 

 

 

 

 

LungsNo. Exam.

No. exam.

50

49

50

50

50

50

49

50

50

50

-  AlvcolarffironchiolarAdenoma

 

 

1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

-  Alveolar/UronchiolarCarcinoma

 

 

 

1

1

 

 

 

 

 

 

 

Metastasis primary siteunknown

 

 

1

 

 

 

 

 

1

 

 

Stomach

No. Exam.

No. exam.

50

49

50

50

50

50

49

50

50

50

- Forestomach Papilloma

 

 

 

 

 

 

 

 

 

 

1

Colon

No. exam.

50

48

50

50

50

50

49

49

49

49

- Fibrosarcoma

 

 

 

 

 

 

 

1

 

 

 

Intestine (residual)&)

No. exam.

 

 

 

 

 

 

 

1

2

2

-Fibroma

 

 

 

 

 

 

 

 

 

1

 

Pancreas

No. exam.

48

49

50

50

50

50

49

50

50

50

- Islet Cell Adenoma

 

1

1

2

3

 

1

 

 

 

 

- Acinar Cell Adenoma

 

 

 

 

 

1

 

 

 

1

 

Thymus

No. exam.

49

49

50

50

50

48

49

50

50

48

- Thymoma (benigne)

 

1

1

 

1

 

 

I

2

 

1

- Thymoma (maligne)

 

1

 

 

 

 

 

 

 

 

 

Mesent. Lymph Node

Mesent. Lymph NodeNo. Exam.

No. exam.

49

449

49

5049

50

50

50

50

50

50

50

50

49

49

50

50

49

49

50

50

- Hemangioma

 

1

1

1

 

1

 

 

 

 

 

Spleen

No. exam.

50

49

50

50

50

50

49

50

49

50

- Hemangiosarcoma

 

1

 

 

 

 

 

 

 

 

 

Skin/Other&)

No. exam.

9

3

5

5

4

6

2

2

2

2

- Schwannoma (mal.)

 

 

1

 

 

 

 

 

 

 

 

- Fibroma

 

 

 

 

1

 

 

 

 

 

 

- Fibrosarcoma

 

1

 

 

 

 

 

1

 

 

 

- Squamous Cell Carcinoma

 

2

1

 

1

 

 

 

 

 

 

- Keratoacanthoma

 

3

 

2

 

1

 

 

 

 

 

- Papilloma

 

 

 

 

 

1

 

 

 

 

 

- Hair Follicle Tumor (ben.)

 

 

 

1

 

 

 

 

 

 

 

-Lipoma

 

 

 

 

 

 

1

 

 

 

 

- Basal Cell Carcinoma

 

 

 

 

 

 

 

 

1

 

 

Mammary Gland/Region

No. exam.

-

-

-

-

-

50

49

50

50

50

-Adenoma

 

 

 

 

 

 

 

1

 

 

 

- Adenocarcinoma

 

 

 

 

 

 

3

1

6

 

2

- Adenocarcinoma arising in a fibroadenoma

 

 

 

 

 

 

1

 

 

 

1

- Fihroadenoma

 

 

 

 

 

 

15

10

7

6

5

- Tumor Mixed (mal.)

 

 

 

 

 

 

1

 

 

1

1

Brain

No. exam.

50

49

50

50

50

50

49

50

50

50

- Astrocytoma (ben.)

 

1

 

 

 

 

 

 

 

 

 

- Granular Cell Tumor (ben.)

 

 

 

 

 

1

 

1

 

 

 

Testes

No. exam.

50

49

50

50

50

-

-

-

-

-

- Leydig Cell Adenoma

 

1

4

 

1

2

 

 

 

 

 

Prostate

No. exam.

50

49

50

50

50

-

-

-

-

-

- Fibrosarcoma

 

 

 

 

1

 

 

 

 

 

 

Ovaries

No. exam.

-

-

-

-

-

50

49

50

50

50

- Granulosa Cell Tumor (ben.)

 

 

 

 

 

 

1

 

 

 

 

- Granulosa Cell Tumor (mal.)

 

 

 

 

 

 

 

 

 

 

1

- Tumor/Sex Cord (ben.)

 

 

 

 

 

 

 

1

1

1

 

Uterus

Uterus

No. exam.

No. Exam.

 

 

 

 

 

50

49

550

50

49

50

50

50

50

-Adenoma

 

 

 

 

 

 

 

 

 

1

 

- Adenocarcinoma

 

 

 

 

 

 

§ 2

2

3

3

8

- Adenosquamous Carcinoma

 

 

 

 

 

 

1

1

1

 

 

- Squamous Cell Carcinoma

 

 

 

 

 

 

 

 

 

 

1

- Schwannoma (mal.)

 

 

 

 

 

 

1

 

1

 

 

- Stromal Sarcoma

 

 

 

 

 

 

 

1

 

1

1

- Stromal Polyp

 

 

 

 

 

 

7

7

4

5

6

- Glandular Polyp

 

 

 

 

 

 

1

 

3

 

 

Vagina

No. exam.

-

-

-

-

-

50

49

50

50

50

- Granular Cell Tumor (ben.)

 

 

 

 

 

 

 

 

 

 

1

Adrenal Glands

No. exam.

50

49

50

50

50

50

49

50

50

50

- Cortical Adenoma

 

 

 

1

 

1

 

1

 

 

 

- Cortical Adenocarcinoma

 

 

 

 

 

 

1

 

 

 

 

- Medullary Tumor (ben.)

 

10

9

5

8

10

1

3

1

1

 

- Medullary Tumor (mal.)

 

2

1

1

1

 

 

 

1

 

 

Pituitary Gland

No. exam.

49

49

50

50

50

50

49

50

50

50

- Adenoma (Pars Distalis)

 

5

6

11

5

8

24

18

24

14

11

- Adenoma (Pars Intermedia)

 

 

 

 

 

 

1

 

 

 

1

- Adenocarcinoma (Pars Distalis)

 

 

 

 

 

 

1

 

1

 

 

Thyroid Gland

No. exam.

50

49

50

50

50

50

49

50

50

50

- C-Cell Adenoma

 

3

7

2

2

3

3

2

3

4

3

- C-Cell Adenocarcinoma

 

1

 

 

 

 

 

 

 

 

 

- Follicular Adenoma

 

#

 

 

1

2

1

1

1

1

1

- Follicular Adenocarcinoma

 

 

 

 

 

1

 

 

 

 

 

Bone/NOS&)

 

 

 

 

 

 

 

 

1

1

 

- Osteosarcoma

 

 

 

 

 

 

 

 

1

 

 

- Schwannoma (maligne)

 

 

 

 

 

 

 

 

 

1

 

Systemic Tumors

No. exam. 

50

49

50

50

50

50

49

50

50

50

- Lymphoma (maligne)

 

2

1

 

2

 

 

 

1

1

 

- Myeloid Leukemia

 

 

1

 

1

 

 

 

 

 

 

- Histiocytic Sarcoma

 

2

1

 

1

1

 

 

 

 

 

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

&) only investigated in case of macro lesions

# positive trend p<0.05

§ positive trend p<0.01

NOS = not otherwise specified

no. = number

exam. = examinations

 

 

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

Sex

 

male

female

Dose ppm

 

0

50

200

1000

3000

0

50

200

1000

4000

Organ/Finding

No. animals

50

50

50

50

50

50

50

50

50

50

 

Body cavities &)

No. exam.

 

5

 

3

 

6

 

6

 

3

 

3

 

3

 

3

 

4

 

6

- Mesothelioma (benign)

 

1

 

 

 

 

 

 

 

 

 

- Lipoma

 

 

 

1

 

 

1

 

 

1

 

- Fibroma

 

 

 

 

 

 

1

 

 

 

 

Clitorial glands &)

No. exam.

 

 

 

 

 

 

1

1

 

 

- Squamous cell carcinoma

 

 

 

 

 

 

 

 

1

 

 

Number of benign tumors

 

29

29

27

23

32

58

47

47

36

33

Malignant tumors

 

15

10

3

7

3

11

8

18

7

16

Total tumors

 

44

39

30

30

35

69

55

65

43

49

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

&) only investigated in case of macro lesions

# positive trend p<0.05

§ positive trend p<0.01

NOS = not otherwise specified

no. = number

exam. = examinations

 

 

 

Table 18: Animals with tumors – terminal kill

Sex

Males

Females

Dose ppm

0

50

200

1000

3000

0

50

200

1000

4000

 

 

lntercurrent Deaths

No. of animals examined

21

19

21

17

13

12

20

24

20

22

Animals with tumors

12

11

13

7

5

10

13

19

12

15

Animals with benign tumors only

5

7

10

3

4

8

7

9

7

5

Animals with malignant tumors only

5

5

3

2

 

1

4

5

2

3

Animals with benign and malignant tumors

2

 

 

2

1

1

2

5

3

7

 

Terminal Kill

No. of animals examined

29

31

29

33

37

38

30

26

30

28

Animals with tumors

17

16

13

16

21

31

20

22

16

15

Animals with benign tumors only

10

11

13

13

19

25

19

15

14

10

Animals with malignant tumors only

2

2

 

2

1

1

 

 

 

1

Animals with benign and malignant tumors

5

3

 

1

1

5

1

7

2

4

 

All Animals

No. of animals examined

50

50

50

50

50

50

50

50

50

50

Animals with tumors

29

27

26

23

26

41

33

41

28

30

Animals with benign tumors only

15

18

23

16

23

33

26

24

21

15

Animals with malignant tumors only

7

7

3

4

1

2

4

5

2

4

Animals with benign and malignant tumors

7

3

 

3

2

6

3

12

5

11

 

 

Table 19: Occurrence of tumor bearing animals in time 

Sex

 

Males

Females

Dose ppm

 

0

50

200

1000

3000

0

50

200

1000

4000

Number of Animals with Neoplasms

 

 

 

 

 

 

 

 

 

 

 

Dying Prematurely

 

12

11

13

7

5

10

13

19

12

15

Neoplasms

benign

11

9

13

7

7

13

11

17

11

14

 

maligne

7

5

3

4

1

3

7

9

5

11

 

total

18

14

16

11

8

16

18

26

16

25

Number of Animals with Tumors

Week

0-13

 

 

1

 

 

 

 

 

 

 

 

 

13-26

 

 

 

 

 

 

 

 

 

 

 

 

27-39

 

 

 

 

 

 

 

 

 

 

 

 

40-52

 

 

 

 

1

 

 

1

1

 

 

 

53-65

 

1

2

 

 

 

 

 

 

2

1

 

66-78

 

1

 

1

1

 

1

2

4

1

2

 

79-91

 

4

2

6

2

2

3

3

8

4

7

 

92-Necropsy

 

6

7

6

3

3

6

7

6

5

5

Conclusions:
CLP: STOT RE 2, H372 Liver
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

Table 1: Mean absolute and relative organ weights

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

* p ≤ 5%

 

Table 2: Gross Pathology Summary #

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)

# : only organs withfindings are shown

 

Table 3: Micropathology summary of tissues # - incidence and average grade

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)

-

-

-

-

-

-

 

inflammation, chronic

-

1(1)

1(2)

-

-

-

-

-

-

-

 

inflammation,granuloma

-

-

-

-

-

-

-

-

-

1(2)

 

inflammation, subacute

-

1(2)

-

-

-

-

-

-

-

-

 

inflammation, suppurative

-

-

-

-

-

-

-

-

-

1(2)

 

macrophage, alveolar

-

-

-

-

2(1.5)

-

-

-

-

-

 

hyperplasia, mesothelium

-

-

-

1(2)

-

-

-

-

-

-

 

microgranuloma

-

-

-

-

-

-

-

-

-

1(1)

Lymph node

no abnormality

4

2

1

2

2

1

1

1

2

4

 

hyperplasia, lymphoid

-

2(2)

3(1.7)

2(2.5)

2(1)

3(1.7

3(1.7)

3(1.3)

1(3.0)

-

 

pigmentation

-

-

-

-

-

-

-

-

1

-

lymph node, mesenteric

no abnormality

4

4

4

3

4

4

4

4

4

3

 

congestion

-

-

-

1(3)

-

-

-

-

-

-

 

hyperplasia, lymphoid

-

-

-

-

-

-

-

-

-

1(2)

Mammary gland

no abnormality

4

4

4

4

4

3

4

4

4

4

 

fibrosis

-

-

-

-

-

1(1)

-

-

-

-

Nasopharynx

no abnormality

4

4

4

4

3

3

4

4

4

4

 

acanthosis

-

-

-

-

-

1(2)

-

-

-

-

 

degeneration

-

-

-

-

1(1)

-

-

-

-

-

Parathyroids

no abnormality

2

3

3

2

3

3

2

4

3

4

 

cyst

2(2.5)

1(2)

1(1)

2(1.5)

1(3)

1(3)

1(2)

-

1(1)

-

 

ectopic tissue

-

-

-

-

-

-

1(1)

-

-

-

Pituitary

no abnormality

3

3

4

3

4

4

4

3

4

2

 

congestion

1(3)

-

-

-

-

-

-

-

-

-

 

cyst

-

1(1)

-

1(1)

-

-

-

1(1)

 

2(1)

Prostate

no abnormality

3

-

3

-

3

-

1

-

4

-

 

cyst

-

-

-

-

1(1)

-

-

-

-

-

 

dilatation

-

-

-

-

-

-

1(2)

-

-

-

 

hyperplasia, cystic

-

-

-

-

-

-

1(2)

-

-

-

 

hypoplasia

-

-

-

-

-

-

1(3)

-

-

-

 

inflammation, chronic

-

-

1(3)

-

-

-

 

-

-

-

 

inflammation, lymphocytic

1(1)

-

-

-

-

-

 

-

-

-

Rectum

no abnormality

4

4

3

4

4

4

4

4

4

4

 

hyperplasia, lymphoid

-

-

1(2)

-

-

-

-

-

-

-

Salivary glands

no abnormality

3

3

2

3

3

4

3

4

2

3

 

inflammation, chronic

-

-

-

1(1)

-

-

-

-

2(1.5)

-

 

inflammation, lymphocytic

1(1)

1(2)

2(1)

-

1(1)

-

1(1)

-

-

1(1)

Small intestine

no abnormality

3

4

4

4

4

2

3

4

3

3

 

cyst

-

-

-

-

-

-

-

-

-

1(3)

 

dilatation, glands

1

-

-

-

-

2(2)

-

-

1(2)

-

 

hyperplasia

-

-

-

-

-

1(2)

-

-

-

-

 

inflammation, chronic

-

-

-

-

-

-

1(3)

-

-

-

Spinal cord

no abnormality

4

3

3

2

2

2

4

4

3

3

 

mineralization

-

1(2)

1(1)

2(1.5)

2(2)

2(2)

-

-

1(2)

1(1)

Spleen

no abnormality

3

4

4

4

4

4

3

3

4

4

 

plaques, fibrosiderotic

1(2)

-

-

-

-

-

1(2)

1(3)

-

-

Stomach

no abnormality

4

4

4

4

3

3

4

4

4

4

 

cyst

-

-

-

-

-

1(1)

-

-

-

-

 

dilatation, glands

-

-

-

-

1(2)

-

-

-

-

-

Testes

no abnormality

4

-

4

-

3

-

3

-

4

-

 

aspermia

-

-

-

-

-

-

1(4)

-

-

-

 

degeneration

-

-

-

-

1(2)

-

-

-

-

-

Third eyelids with glands

no abnormality

4

3

4

4

4

4

4

4

4

4

 

hyperplasia, lymphoid

-

1(3)

-

-

-

-

-

-

-

-

Thymus

no abnormality

3

4

1

3

2

2

3

4

3

2

 

involution

1(3)

-

3(1.7)

1(1)

2(2.5)

2(1.5

1(4)

-

1(1)

2(3)

Thyroids

no abnormality

1

1

2

-

3

2

-

1

2

1

 

colloid reduced

-

-

1(2)

-

-

-

-

-

-

1(3)

 

degeneration

1(3)

1(1)

1(1)

1(2)

-

-

2(1.5)

1(2)

1(3)

-

 

inflammation, chronic

-

-

-

-

-

-

-

1(2)

-

-

 

inflammation, lymphocytic

-

-

-

-

-

-

1(3)

1(3)

-

3(3)

 

hyperplasia, c-cell

2(2)

3(2)

1(3)

4(1.5)

1(2)

2(2)

4(1)

2(2.5)

2(1.5)

2(2.5)

Trachea

no abnormality

4

3

4

4

4

4

4

4

4

4

 

hyperplasia, epithelial cell

-

1(2)

-

-

-

-

-

-

-

-

() : Average severity of animals with lesions: 1 (minimal) to 5 (severe)

* : significantly different from control (p=≤0.05)

- : no incidence

m: male f: female

# : only tisues with micoropathology findings are shown

 

Table 4: Special hepatocellular enzymes - mean

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*

* P ≤ 5%

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

Table 1: Body weight (g) - main groups

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

* statistical evaluation not possible

+ = 5 % significance level

 

Table 2: Body weight (g) - recovery groups

dose (mg/kg)

day 0

day 7

day 14

day 21

day 28

day 35

day 42

sex

0

259

271

278

290

295

320

319

male

1000

261

278

294

305

321

342

339

male

0

227

229

233

236

241

247

239

female

1000

227

227

231

233

242

243

237

female

+ = 5 % significance level

 

Table 3: Mean feed intake

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

* statistical evaluation not possible

+ = 5 % significance level

 

Table 4: Mean feed intake – recovery groups

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

+ = 5 % significance level

 

Table 5: Hematology

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)

+ = 5 % significance level

++ = 1 % significance level

 

Table 6: Activity cholinesterase

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)

+ = 5% significance level

++ = 1 % significance level

Table 7: Absolute and relative organ weights

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)

+ = 5 % significance level

 

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

Table 1: Body weight (g) - main groups

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

* statistical evaluation not possible

+ = 5 % significance level

 

Table 2: Body weight (g) - recovery groups

dose (mg/kg)

day 0

day 7

day 14

day 21

day 28

day 35

day 42

sex

0

259

271

278

290

295

320

319

male

1000

261

278

294

305

321

342

339

male

0

227

229

233

236

241

247

239

female

1000

227

227

231

233

242

243

237

female

+ = 5 % significance level

 

Table 3: Mean feed intake

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

* statistical evaluation not possible

+ = 5 % significance level

 

Table 4: Mean feed intake – recovery groups

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

+ = 5 % significance level

 

Table 5: Hematology

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)

+ = 5 % significance level

++ = 1 % significance level

 

Table 6: Activity cholinesterase

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)

+ = 5% significance level

++ = 1 % significance level

Table 7: Absolute and relative organ weights

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)

+ = 5 % significance level

 

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.

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, gross pathology examinations, determination of organ weights and histopathology analysis.

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