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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
chronic toxicity: oral
Remarks:
Combined carcinogenicity study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 Jan 1990 to 24 Jan 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:
1981
Qualifier:
according to guideline
Guideline:
other: FIFRA, Pesticide Assessment Guidelines, subdivision F:Hazard Evaluation, Human and Domestic Animals, Section 83-5: Combined chronic toxicity/oncogenicity studies
Version / remarks:
1982
Qualifier:
according to guideline
Guideline:
other: Agricultural Production Bureau, Ministry of Agriculture, Forestry and Fisheries, Japan, 59 Noh San No. 4200, dated January 28, 1985: "Guidance on Toxicology Study data for Application of Agricultural Chemical Registration".
Version / remarks:
1985
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)-phenyl-aminocarbonyl]-2,6-difluorobenzamide
EC Number:
410-690-9
EC Name:
N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)-phenyl-aminocarbonyl]-2,6-difluorobenzamide
Cas Number:
103055-07-8
Molecular formula:
C17 H8 Cl2 F8 N2 O3
IUPAC Name:
1-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)phenyl]-3-(2,6-difluorobenzoyl)urea

Test animals

Species:
rat
Strain:
other: Tif: RAIf (SPF), hybrids of RII/l x RII/2 (Sprague-Dawley derived)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: Approx. 5 weeks
- Weight at study initiation: 99.35 - 139.8 g in males and 87.80 - 130.7 g in females
- Housing: The animals were housed in groups of 5 in Macrolon cages type 4 with standardised granulated soft wood bedding
- Diet: Standard diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 10
- Air changes (per hr): 16 to 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 22 Jan 1990 to 24 Jan 1992

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet: Fresh diets were prepared at 4 week intervals throughout the study.
- Mixing appropriate amounts with type of feed: The test substance was weighed on a calibrated Mettler balance. The pulverised food was then homogeneously mixed with the appropriate concentrations of the test substance and about 25 %water was added before pelleting to ensure the necessary pellet quality. The pellets were subsequently airdried and stored at room temperature until used. The animals in the control group (group 1) were fed with
similarly pelleted food without the test substance.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Food samples were analysed for concentration, homogeneity and stability. Analysis of the diet concentration were undertaken periodically with the diet batches applied during the study.
Duration of treatment / exposure:
24 months
Frequency of treatment:
Continuously
Doses / concentrationsopen allclose all
Dose / conc.:
5 ppm
Remarks:
Group 2: Dietary equivalent to 0.192 and 0.229 mg/kg bw/day for males and females, respectively
Dose / conc.:
50 ppm
Remarks:
Group 3: Dietary equivalent to 1.93 and 2.34 mg/kg bw/day for males and females, respectively.
Dose / conc.:
500 ppm
Remarks:
Group 4: Dietary equivalent to 20.4 and 24.8 mg/kg bw/day for males and females, respectively.
Dose / conc.:
1 500 ppm
Remarks:
Group 5: Dietary equivalent to 108 and 114 mg/kg bw/day for males and females, respectively. Due to severe toxicity, all group 5 animals were sacrificed in week 14
No. of animals per sex per dose:
80 animals per sex per dose were used, consisting of the following Experimental groups:
Experimental group I: 50 animals/sex/group for evaluation of the carcinogenic potential of the test substance.
Experimental group II: 10 animals/sex/group for haematological, biochemical and urine analysis and sacrifice after 24 months.
Experimental group III: 10 animals/sex/group for haematological investigations and sacrifice after 24 months.
Experimental group IV: 10 animals/sex/group for interim sacrifice at month 12.
Control animals:
yes, plain diet

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: Daily, records at least weekly (in life observations) and twice daily on working days and once a day on weekends and holidays (mortality)

BODY WEIGHT:
- Time schedule for examinations: Weekly (midweek) for the first 3 months and monthly thereafter

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
- Time schedule: Weekly for the first 3 months and monthly thereafter.

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:
- Time schedule: monthly

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule for examinations: Animals of the control and highest dose level (exp. group I) were examined before the beginning of treatment. At 6, 12 and 18 months control and group 4 animals (due to early demise of group 5 animals) were examined. At 24 months, surviving animals of all dose levels of group were examined.

HAEMATOLOGY:
- Time schedule for collection of blood: Week 13, 26, 53, 78 and 105
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: 20 animals of each sex and group (experimental groups II and III). For terminal laboratory investigations at week 105, the number of animals subjected to examination was fortified by animals of the carcinogenicity group (I) and/or experimental group III to obtain 20 samples/sex/group for haematology
- Parameters checked: Erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean leukocyte count, mean leukocyte differential count, thrombocyte count

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: Week 13, 26, 53, 78 and 105
- Animals fasted: Yes
- How many animals: 10 animals per sex per group (experimental group II). For terminal laboratory investigations at week 105, the number of animals subjected to examination was fortified by animals of the carcinogenicity group (I) and/or experimental group III to obtain 10 samples/sex/group for blood chemistry.
- Parameters checked: Glucose, urea, creatinine, total bilirubin, total protein, albumin, globulins, a/g ratio, cholesterol, sodium, potassium, calcium, chloride, aspartate aminotransferase, amino transferase, alkaline phosphatase, gamma-glutamyl

URINALYSIS:
- Time schedule for collection of urine: Urine for analysis was collected overnight.
- Metabolism cages used for collection of urine: Yes
- Parameters checked: Urine volume, relative density, pH-value, urine color, protein, glucose, ketones, bilirubin, blood, urobilinogen
- Microscopic examination: cells, casts, crystals

Sacrifice and pathology:
GROSS PATHOLOGY:
- After one and two years of treatment, the control and treated animals scheduled for each sacrifice were bled under ether anesthesia and subjected to detailed necropsy. At necropsy following weights were for these animals: Body (exsanguinated), brain, liver, kidneys, adrenals and gonads (weights)
- The following organs and tissues of these animals were preserved in neutral buffered 4% formalin: Skin, mammary area, spleen, mesenteric lymph node, axillary lymph node, popliteal lymph node, sternum with bone marrow, femur with joint, skeletal muscle, trachea, lung, heart, aorta, submandibular salivary gland, both, liver, pancreas, stomach, small intestine large intestine, kidney, both, urinary bladder, prostate, seminal vesicle, testis, both, epididymis, both, uterus, vagina, ovary, both, pituitary gland, adrenal gland, both, thyroid with parathyroid gland, thymus, peripheral nerve, brain, spinal cord, eye with optic nerve, both orbital gland, both, extraorbital lacrimal gland, both, zymbal gland, both, muzzle, tongue tissues with gross lesions. Carcass and organ weights were not recorded for these animals.

HISTOPATHOLOGY: Skin, mammary area, spleen, mesenteric lymph node, axillary lymph node, popliteal lymph node, sternum with bone marrow, femur with joint, skeletal muscle, trachea, lung, heart, aorta, submandibular salivary gland, both, liver, pancreas, esophagus, stomach small intestine, large intestine, kidney, both, urinary bladder, prostate, seminal vesicle, testis, both epididymis, both, uterus, vagina, ovary, both, pituitary gland, adrenal gland, both, thyroid with parathyroid gland, thymus, peripheral nerve, brain, spinal cord, eye with optic nerve, both, orbital gland, both, extraorbital lacrimal gland, both, Zymbal gland, both, muzzle, tongue and tissues with gross lesions

OTHER
Sacrifice:
- Unless prevented by advanced autolysis, cannibalism or by technical reasons, a complete necropsy with tissue preservation was performed also on all animals which died during the test period or which had to be sacrificed in moribund condition as well as on all males and females of group 5 (1500 ppm) which were sacrificed after 3 months of treatment. Carcass and organ weights were not recorded for these animals.
- A scheduled interim sacrifice of 10 animals per sex and dose group was performed at 12 months (group IV)
- Sacrifice after 24 months: Group I, II and III

Other examinations:
- Blood levels and residues in fat:
These parameters were determined from all 1 year interim sacrifice animals (IV) and from all surviving animals of experimental group III, samples of fat (approximately 5 g) and blood (approximately 5 mL) were obtained at necropsy. Group blood and fat samples were pooled according to sex, deep frozen and forwarded to the analytical laboratory
Statistics:
For each time point and parameter a univariate statistical analysis was performed. Nonparametric methods were applied, to allow for non normal as well as normal data distribution. Each treated group was compared to the control group by Lepage’s two-sample test and tested for
increasing or decreasing trends from control up to the respective dose group by Jonckheere’s test for ordered alternatives. The Lepage test is a combination of Wilcoxon and Ansari-Bradley statistics, i.e. a combined test for location and dispersion. The Lepage test has a good power against the more general alternative that the distributions differ not only in location but also in dispersion. The Jonckheere test is sensitive to monotone dose-related effects. Survival analysis was performed by the regression model (partial likelihood) introduced by Cox in order to compare survival time of treated animals with control animals. Statistical significance does not necessarily imply biological relevance. Hence, the responsible scientist may not comment on statistically significant values lying within the physiological range and on the other hand may comment on values, which differ substantially from the expected normal values although this difference was not statistically significant.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
A total of males and 57 females in group 5 (1500 ppm) and males and 58 females in group (500 ppm) exhibited whole body tonic- clonic convulsions from weeks 6 and 7, respectively.
Convulsive episodes lasting approximately 30 to 90 seconds were observed mainly during and after handling, and occurred repeatedly over several weeks in the majority of affected animals the study progressed the incidence of observed convulsions in group 4 diminished.
Within this colony of rats, a low frequency of spontaneous convulsive episodes has previously been observed in untreated rats. In the present study, 7 males and 2 females of the control group were seen to convulse. The convulsions recorded among controls and rats of groups 2 and 3 (5 and 50 ppm) were of a shorter duration and less intense than those recorded in groups 4 and 5 (500 and 1500 ppm), and are, therefore, regarded as spontaneous events.
A higher incidence of reddened and/or swollen eyelids, often associated with eye exudate (males and females group 4) or swollen eyelids (males group 3) was recorded. This was a phenomenon occurring unilaterally or bilaterally, and usually resolving after several weeks. The etiology of this change is unknown although both unilaterality and transient occurrence suggest an external irritation, which upon scheduled ophthalmological examinations was observed only at minimal incidences. Therefore, this finding was neither considered a direct response to treatment nor an adverse effect.
In females of group 4 (500 ppm) a high incidence of animals with vaginal discharge was recorded towards the end of the treatment period although no common pathology was detected.
Mortality:
mortality observed, treatment-related
Description (incidence):
Due to the overt toxicity recorded for animals of group 5 (1500 ppm) all males and females of this group were sacrificed in week 14. Survival among the remaining groups was not disturbed by treatment
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Lower mean body weights were recorded for males and females of group 5 (1500 ppm) which by week 12 (prior to early sacrifice) were approximately 10% lower than control values.
Mean body weights for males of group 4 (500 ppm) were significantly lower than control values from 7 onwards, attaining a maximum difference of 8%.
Slightly lower mean body weights were recorded for both sexes in the 500 ppm group up to week 27. Thereafter, an increased body weight gain in the females resulted in mean values approximately 20 % higher than controls by the end of the study. The males in the 500 ppm group continued to have body weights slightly lower than controls to the end of the study.
Slightly higher mean body weights for group 2 females (5 ppm) were not dose-related and considered incidental. Therefore, it was concluded that treatment had not influenced body weight gain in groups 2 and 3 (5 and 50 ppm)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
For the first week of treatment, food intakes by males and females of groups 4 and 5 (500 and 1500 ppm) were 11 to 14% higher than control values. Thereafter the food intake of the 1500 ppm group tended to be lower than controls up to the early termination of this group. Lower food intake compared to controls was recorded in the 500 ppm males for the next 26 weeks after which time the values were similar to controls. Females in the 500 ppm group showed an 18% increase in food intake during weeks 23 to termination. The overall intakes (totals of weeks 1-103) by groups 2 and 3 (5 and 50 ppm) were essentially similar to those of control group.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Water intake by males and females was not influenced by treatment with the test susbtance, apart from transiently higher intakes recorded during weeks 16-28 for females in the 500 ppm group.
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No conclusive evidence of a treatment-related effect of the test substance on the eyes. xaminations included inspection of the surroundings of the eyes, of sclera, cornea, iris and adaptation of the pupil to the ophthalmoscopic light beam. Animals of group 1 (control) and group 5 (1500 ppm) were examined at the beginning of the study (day -5). Due to the early demise of group 5, control animals and group 4 (500 ppm) were examined during days 171, 360 and 543 of the treatment period. During day 723, animals of group 1 (control), group 2 (5 ppm), group 3 (50 ppm) and group 4 (500 ppm) were examined.
The eye examinations gave no conclusive evidence of an effect of treatment although on most occasions a small number of treated rats showed reddened or swollen eyelids, eye exudate, and ptosis of eyelids. However, the incidence was not statistically significant and within the expected range. Therefore, this finding was not considered of toxicological relevance.
The number of animals with cloudy/opaque eyes (eye(s) and/or colour altered) and associated absent pupil reflex appeared higher in females of groups 2 and 4 (5 and 500 ppm) at day 723. However, reference to the in-life observations recorded on a continuous basis, as opposed to one day in isolation, shows a similar incidence of this change across all groups. Therefore, it is concluded that the eyes of the rats were not affected by treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The week 13 examination one week prior to the termination of group 5 (1500 ppm) showed 8/20 males with platelet counts above the concurrent control range and 3/20 females with white blood cell counts above the control range. Other parameters in group 5 animals were not influenced by treatment. Subsequent examinations revealed no evidence of a treatment-related influence on the haematological profile of treated rats. White blood cell counts obtained for one female (control group), diagnosed with blast cell leukemia, unduly influenced the group mean value at week 105. A number of differences between the means attained a level of statistical significance. However, the difference were of a small order of magnitude and considered to be of no biological relevance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Treatment did not influence the blood chemistry profile of male and female rats of groups 2, 3 and 4 (5, 50 and 500 ppm). Slightly lower values for plasma protein and albumin and higher levels of plasma potassium and inorganic phosphorus were recorded for group 5 females prior to the termination of this group in week 14.
A number of differences between the means attained a level of statistical significance. However, the differences were of a small order of magnitude and considered to be of no biological relevance.
Endocrine findings:
not examined
Urinalysis findings:
no effects observed
Description (incidence and severity):
The quantitative and qualitative tests performed gave no indication of a treatment related effect on renal function
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):
Compared with the controls, levels of statistical significance were achieved for adrenal gland ratios for females of groups 3 and 4 (50 and 500 ppm) sacrificed at week 53. However, no changes were detected in the adrenals by microscopy and the ratios at termination were similar to control values. Therefore, the difference at week 53 was considered to have occurred by chance. Other differences which attained a level of statistical significance were of a small order of magnitude and not associated with histopathological change. Therefore, these differences were considered to be of no biological relevance
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Higher incidences of mottled lungs in males and females of group 4 (500 ppm) were considered to be treatment-related. The microscopical verification of some other macroscopical findings reported in higher numbers of treated animals revealed no treatment-related gross pathological effects.
Macroscopically reported "small" seminal vesicles in 7/80, 6/80, 4/80, and 14/80 males of groups 1, 2, 3, and 4, respectively, (0, 5, 50, 500, and 1500 ppm) did not show any pathological change on microscopical examination.
Mottled thymus was reported at necropsy in 14/80 males and 11/80 females of group 5 (1500 ppm). In 8 out of those 14 males and 6 out of the 11 females no microscopical changes were observed. The remaining 6/80 (=7.5%) males and 5/80 (=6.3%) females showed minimal to moderate recent haemorrhage in the thymus. In the control groups (0 ppm) 5/77 (=6.5%) males and 3/78 ( =3.8%) females presented with the same lesion. The slight increase in the incidences of thymic haemorrhage in group 5 was considered most likely related to the convulsions reported in this group and not as a direct result of treatment.
Scab formation on the skin of the tail found in 7/80 females of group 4 (500 ppm) and 1/80, 1/80, 11/80, and 4/80 males of groups 2, 3, 4, and 5, respectively, (5, 50, 500, and 1500 ppm) Here found to be related to tail injuries (bite marks) seen in higher numbers of animals of groups 4 and 5 suffering from tonic-clonic convulsions.
Large adrenal glands were reported in 2/80, 5/80, 5/80, and 8/80 males and 3/80, 4/80, 2/80, and 11/80 females of groups 1, 2, 3, and 4, respectively, (0, 5, 50, and 500 ppm). On microscopical examination various lesions, such as cysts, sinusoidal cystic dilatations, and hyperplasia’s or tumours of cortex or medulla, were found in these adrenals. The incidences of these lesions in the different groups did not indicate any treatment-related effect.
Compared with the control group, a decrease in the number of animals bearing masses and/or nodules as well as in the total number of masses and/or nodules, especially those found on the body surface, was observed in group 4 (500 ppm), particularly in females. In group 5 (1500 ppm) which was sacrificed after 3 months of treatment no masses or nodules were found.
The majority of these masses correlated microscopically with proliferative, hyperplastic or neoplastic lesions which were also decreased in number in the animals of group 4. All other macroscopical findings occurred in comparable numbers in all experimental groups. They were similar to those occurring spontaneously in our colony of rats and are, therefore, considered to be incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Lung: Aggregations of alveolar foam cells were observed in higher numbers in males and females of groups 4 and 5 (500 and 1500 ppm). Additionally, the severity of this lesion was found to be increased in animals of group 4 surviving the 2 year test period but not in the animals of group 5 which were sacrificed after 3 months of treatment.
Heart: Secondarily, increased pulmonary pressure caused by massive occupation of alveolar lumen by foam cells and impairing the haemodynamics in some female animals resulted in a dilatation of the right heart ventricle without any treatment-related change of the heart muscle itself. This change was found to be increased in incidence in female group 4 (12/80) when compared to the control and other treated groups (4/80, 1/80, and 4/80 females in groups 1, 2, and 3, respectively).
Nonglandular stomach: In the nonglandular stomach, ulcerative and inflammatory lesions were found to be increased in number in group 4 (500 ppm).
Large intestine: Focal hemorrhagic, necrotic (infarct), ulcerative, and inflammatory changes of the large intestine (cecum and/or colon) were found at higher incidences in males and females of groups 4 and 5 when compared with the control and other treated groups. These changes, belonging to the same lesion complex.
Liver: In group 4 (500 ppm), fatty change in the perilobular region of the liver was seen in 36/80 females. Such hepatic fatty change was not observed in group 5 (1500 ppm) which was sacrificed after three months of treatment. In comparison, only 2/80 control females, 2/80 females of group 2 (5 ppm) and 1/80 females of group 3 (50 ppm) presented this lesion. In males, the incidences of this lesion did not indicate a dose-related effect.
Urinary tract: A most likely ascending inflammation of the female urinary tract consisting of chronic inflammation of the urinary bladder, chronic inflammation of the renal pelvis, and pyelonephritis, was found to be markedly increased in incidence at 500 ppm.
Skin: Ulcerative and inflammatory lesions in the skin were noticed in higher numbers of males of groups 4 and 5 and females of group 5. The majority of these lesions correlated with macroscopically reported scab formations on the skin of tail which were found to be related to tail injuries (bite marks) seen in higher numbers of animals of groups 4 and 5 suffering from tonic-clonic convulsions. Such lesions were not considered as a direct result of treatment.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Proliferative lesions: No treatment-related increase of hyperplastic or neoplastic lesions was found. Some hyperplastic and neoplastic lesions were found in decreased numbers in animals of group 4, especially those of the adenohypophysis in males and of the mammary gland in females. No proliferative changes were found in group 5 which was sacrificed after 3 months of treatment. In the statistical analysis the benign interstitial cell tumour of testis found at incidences of 2/80 in group 1, 2/80 in group 2, 1/80 in group 3, and 5/80 (6.25%) in group 4, as well as the benign granular cell tumour of the cerebral meninges found in male animals at incidences of 1/80 in group 1, 0/80 in group 2, 1/80 in group 3, and 3/80 (3.75%) in group 4 showed an apparent statistical evidence for a dose-related effect in group 4. Nevertheless, these incidences were within the range of historical control data and were, therefore, considered as incidental.
Other effects:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
50 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
food consumption and compound intake
haematology
histopathology: non-neoplastic
Remarks on result:
other: Corresponding to an average daily intake of 1.93 and 2.34 mg/kg bw/day in males and females, respectively

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Analytical results: The results indicate that the test substance mixed with rodent diet is stable for at least 35 days at room temperature. Furthermore, the diet preparation procedure yields homogeneous diets.

Table 1. Results test substance intake (mg/kg bw/day) in rats (corrected for purity)

 

Dietary concentration (ppm)

Group 2

5

Group 3

50

Group 4

500

Group 5

1500

Males

0.192

1.93

20.4

108

Females

0.229

2.34

24.8

114

Table 2. Clinical signs and mortality in rats treated with the test substance

 

Males

Females

0

5

50

500

1500

0

5

50

500

1500

In-life findings

Convulsions

7

1

5

47

46

2

6

4

58

57

Tail, lesion/injured

1

1

1

14

23

0

0

0

19

5

Vaginal discharge

-

-

-

-

-

0

0

0

18

0

Mortality

No. of survivors to terminal necropsy*

35

32

40

41

0

36

40

41

43

0

% survivors

50

46

57

59

-

51

57

59

61

-

*survival of originally 70 animals/sex/group (Experimental groups I-III)

Table 3. Body weights of rats treated with the test substance

 

Males

Females

0

5

50

500

1500

0

5

50

500

1500

Week Body weights (g)

-1

115.7

119.0

117.2

116.7

119.9*

110.6

108.6

108.7

110.2

107.9

1

168.5

172.1

168.0

167.7

169.3

144.2

142.3

141.8

143.0

140.3*

4

315.8

317.0

308.5

311.1

303.3*-

217.6

220.8

218.1

214.9

203.4*-

5

352.0

352.0

343.2

344.2

335.6*-

233.8

236.6

232.2

228.2

213.7*-

6

377.4

377.9

368.2

369.8

359.1*-

244.9

250.3

246.6

238.9*

225.1*-

7

400.5

399.9

391.8

389.3-

376.9*-

256.7

261.4

257.1

245.3*-

230.8*-

8

417.3

416.3

406.1

403.4*-

392.3*-

265.7

268.6

264.6

250.8*-

237.1*-

9

437.0

434.8

424.2

419.6*-

404.6*-

268.2

278.1

271.1

258.6-

241.9*-

10

450.6

450.3

437.9

432.8*-

415.9*-

278.4

285.2

277.7

265.5*-

247.6*-

11

467.8

465.6

453.1

446.8*-

428.5*-

282.8

288.0

283.6

266.7*-

256.5*-

12

481.1

478.3

464.8

457.8*-

436.5*-

288.1

295.6

287.2

269.6*-

259.3*-

15

516.3

511.0

496.2-

489.3*-

 

300.7

308.6

300.8

282.7*-

 

27

612.5

605.8

591.9

563.6*-

 

329.8

349.5+

335.8

326.5

 

39

678.2

673.5

655.8

637.2*-

 

350.9

376.0*+

359.3

403.7*+

 

55

756.2

745.5

734.1

711.6*-

 

384.7

419.6+

401.6

484.4*+

 

71

819.4

818.5

798.6

772.2*-

 

431.1

473.9+

443.9

530.0*+

 

87

834.4

832.6

826.0

796.6

 

466.0

502.8

470.6

550.6*+

 

103

790.2

761.0

754.3

728.9

 

442.7

493.7

446.4

535.3*

 

*: statistically significantly different from control, p<0.01 (Lepage)

+-: statistically significantly different from control, p<0.01 (Jonckheere)

Table 4. Food consumption of rats treated with the test substance

 

Males

Females

0

5

50

500

1500

0

5

50

500

1500

Week Food consumption (g/animal/week)

-1

116.9

119.5

116.8

117.9

120.1

102.8

101.7

102.5

101.9

100.4

1

138.8

144.6

140.8

153.5*+

157.6*+

107.3

106.9

110.1

119.6*+

119.3*+

4

185.9

187.6

182.3

185.7

179.1*

132.2

136.7

134.2

131.6

120.9*-

5

189.6

194.1

175.0*-

181.5-

179.5*-

129.4

138.0*+

118.5*

120.7*-

116.1*-

6

188.7

188.1

176.3*-

181.1*-

177.0*-

129.4

131.2

128.6

124.4

116.4*-

7

187.6

184.4

174.7*-

176.6*-

169.5*-

130.3

132.4

127.0

121.0*-

115.0*-

8

187.7

184.9

176.8*-

174.1*-

167.4*-

127.7

131.3

125.9

119.5*-

114.0*-

9

178.4

183.3

171.0

178.0

175.5

116.3

128.0*+

120.6

127.0*+

121.4

10

178.9

182.8

171.1

176.2

176.3

119.0

121.1

117.3

118.1

119.2

11

184.5

184.7

172.3*-

176.5-

173.2*-

119.6

121.3

116.8

113.4

115.5

12

181.3

181.9

171.2-

171.6*-

169.9-

122.2

127.6

120.7

113.9-

121.1

15

180.1

175.0

171.7-

160.6*-

 

115.4

118.9

115.2

111.6

 

27

173.1

168.0

164.4

164.9-

 

108.6

111.7

112.3

129.2*+

 

39

168.9

170.7

168.7

169.1

 

112.0

119.0

114.7

141.3*+

 

55

188.2

184.9

184.1

183.8

 

128.7

132.8

125.0

148.7

 

103

151.3

156.5

149.9

143.4

 

118.8

126.2

119.6

138.9*

 

*: statistically significantly different from control, p<0.01 (Lepage)

+-: statistically significantly different from control, p<0.01 (Jonckheere)

Table 5. Haematological paramterters at week 13

 

Males

Females

0

5

50

500

1500

0

5

50

500

1500

Haematology

Platelet count

1060

1104

1091

1038

1242*

1031

1048

1137+

1038

1116

White blood cell

count

13.45

12.48

13.13

11.73

12.04

8.186

7.503

7.429

8.223

10.25+

Blood chemistry

Protein

70.16

71.99

71.59

71.17

70.55

70.67

71.35

71.51

71.25

68.86

Albumin

37.90

37.89

37.54

38.07

37.34

40.09

39.70

39.71

39.09

38.08

Potassium

3.511

3.359

3.472

3.616

3.583

3.183

3.236

3.054

3.094

3.623*

Phosphorus

1.692

1.646

1.739

1.687

1.782

1.483

1.443

1.406

1.503

2.039*+

* Statistically significant difference from control group mean, p<0.01 (Lepage's test)

+ Statistically significant difference from control group mean, p<0.01 (Jonkheere's test)

Table 6. Organ weights and organ to body weight ratio

 

Males

Females

0

5

50

500

0

5

50

500

Organ weights

Abs.adrenals w53

70.59

82.67

74.23

82.93

79.86

92.69

99.00*

111.6*+

w105

116.2

130.1

192.5

129.2

102.3

123.3

100.7

137.4*+

Rel. adrenal w53

0.107

0.116

0.118

0.131

0.204

0.251

0.270+

0.266+

w105

0.164

0.188

0.284

0.205

0.261

0.291

0.250

0.293

* Statistically significant difference from control group mean, p<0.01 (Lepage's test)

+ Statistically significant difference from control group mean, p<0.01 (Jonkheere's test)

Table 7. Macroscopical findings

 

Males

Females

0

5

50

500

1500

0

5

50

500

1500

Initial no. of rats

80

80

80

80

80

80

80

80

80

80

Mottled lungs

Mottled lungs

7

7

5

17

0

13

8

8

26

0

Ratio %

9

9

6

21

0

16

10

10

33

0

Masses & nodules

No. of rats with masses

and/or nod.

40

38

41

36

0

46

57

49

33

0

Ratio %

50

48

51

45

0

58

71

61

41

0

Total no. of masses and/or nodules:

- In the whole body

70

54

60

44

0

99

128

92

42

0

- On the body surface

39

34

30

22

0

84

110

78

29

0

Table 8. Inflammation of the female urinary tract

 

Females

0

5

50

500

1500

Initial no. of rats

80

80

80

80

80

examined urinary tract

80

80

80

80

80

With inflammation of the urinary tract

4

5

3

26

0

Ratio (%)*

5

6

4

33

0

examined urinary bladder

79

80

79

79

77

chronic inflammation of the urinary bladder

1

2

2

22

0

examined kidney

80

80

80

80

80

chronic inflammation of the renal pelvis

2

4

2

20

0

pyelonephritis

1

0

0

3

0

Table 9. Microscopical findings

 

Males

Females

0

5

50

500

1500

0

5

50

500

1500

Initial no. of rats

80

80

80

80

80

80

80

80

80

80

Foam cells in the pulmonary alveoli

No. of rats w/ foam c.

37

40

34

54

67

43

43

43

65

74

Ratio (%)

46

50

43

68

84

54

54

54

81

93

Lesion graded as:

Minimal (+)

20

24

19

24

61

21

25

21

22

69

Ratio (%)

54

60

56

44

91

49

58

49

34

93

Moderate (++)

12

12

10

19

6

14

15

16

24

5

Ratio (%)

32

30

29

35

9

33

35

37

37

7

Marked (+++)

5

4

5

11

0

8

3

6

19

0

Ratio (%)

14

10

15

20

0

19

7

14

29

0

Ulcerative and inflammatory lesions in the nonglandular stomach

Examined nongl stom.

80

80

79

79

80

80

79

80

79

80

Ulcer. and infl. lesions

3

0

2

8

0

3

4

3

13

0

Ratio (%)

4

0

3

10

0

4

5

4

16

0

Lesion described as:

Ulceration

0

0

1

4

0

1

0

1

2

0

Inflammatory oedema

0

0

0

0

0

1

0

1

0

0

Inflammatory cell

infiltration

1

0

0

0

0

0

0

0

0

0

Chronic inflammation

2

0

1

4

0

1

4

1

11

0

Focal haemorrhagic, necrotic ulcerative, and inflammatory lesions in the large intestine

Examined large intest.

78

79

79

78

80

79

79

80

80

80

With lesions in large

intestine*

0

0

0

5

10

1

1

1

5

6

Ratio (%)

0

0

0

6

13

1

1

1

6

8

Examined cecum

78

78

78

76

80

79

79

80

80

80

Lesions in the cecum*

0

0

0

2

8

1

0

1

3

6

Lesion described as:

Haemorrhage

0

0

0

0

2

0

0

0

0

0

Hemorrhagic infarct

0

0

0

0

0

0

0

0

0

4

Ulceration

0

0

0

0

0

0

0

0

0

1

Inflammatory oedema

0

0

0

0

7

0

0

0

0

1

Inflammatory cell

infiltration

0

0

0

1

1

0

0

1

2

0

Chronic inflammation

0

0

0

1

0

0

0

0

1

0

granuloma

0

0

0

0

0

1

0

0

0

0

Examined colon

78

79

79

78

80

79

79

79

80

80

Lesions in the colon*

0

0

0

3

2

0

1

0

4

1

Lesion described as:

Haemorrhagic infarct

0

0

0

0

0

0

0

0

0

1

Ulceration

0

0

0

1

0

0

1

0

0

0

Inflammatory oedema

0

0

0

0

1

0

0

0

0

0

Inflammatory cell infiltration

0

0

0

0

1

0

0

0

2

0

Chronic inflammation

0

0

0

0

0

0

0

0

1

0

Fibrosis

0

0

0

2

0

0

0

0

1

0

Historical data proliferative lesions:

Benign interstitial cell tumour in the testis of untreated male rat in 18 24-month studies conducted

between 1978 and 1989:

• mean 2.72% [37/1361]; SD: 0.02;

• range high: 7.50% [6/80];

• range low: 0.00% [0/80].

Benign granular cell tumour in the meninges of untreated male rats in 18 24-month studies

conducted between 1978 and 1989:

• mean 1.47% [20/1357]; SD: 0.02;

• range high: 5.00% [4/80];

• range low: 0.00% [0/80].

Applicant's summary and conclusion

Conclusions:
It was concluded that the NOAEL was 50 ppm, corresponding to an average daily intake of 1.93 and 2.34 mg/kg bw/day in males and females, respectively. The test substance showed no potential for carcinogenic effects in rats.
Executive summary:

In an OECD TG 453 study in compliance with GLP, the test substance was administered in the diet at dietary levels of 0, 5, 50, 500 and 1500 ppm for 24 months to a total of 800 albino rats, 80 males and 80 females per dose group. A scheduled interim sacrifice of 10 animals per sex and dose group was performed at 12 months. Average achieved intakes corrected for the quantity of the test substance by frequent chemical analysis were 0.19, 1.93, 20.4 and 108 mg/kg bw/day for males and 0.23, 2.34, 24.8 and 114 mg/kg bw/day for females, of group 2, 3,4 and 5, respectively. Achieved intakes for group 5 relate to their 14 week treatment period. Food samples were analysed for concentration, homogeneity and stability. Analysis of the diet concentration were undertaken periodically with the diet batches applied during the study.

Results showed whole body tonic-clonic convulsions occurred with earliest onset at week 6 in the majority of males and females of groups 4 and 5 (500 and 1500 ppm). In several of these animals, bite marks on the tail were present. Furthermore, several group 4 females had vaginal discharge during the latter part of the treatment period. The convulsive episodes observed in group 5 (1500 ppm) led to the decision to terminate treatment and sacrifice the group in week 14. The mortality among the remaining treated groups was similar to that of the controls. The males and females of group 5 (1500 ppm) in the week 12 measurement (prior to early sacrifice) revealed body weights approximately below control values. Lower mean body weights were recorded for males and females of group 4 (500 ppm) to week 27. Thereafter, an increased gain by females resulted in mean values approximately 20% higher than controls by the end of the study. Lower mean body weights continued to be recorded for group 4 males. Higher intakes were recorded in week 1 for males and females of groups and 5 (500 and 1500 ppm). Thereafter, group 5 intakes tended to be lower than control values to the early termination of this group. Lower intakes were also recorded for males of group 4 (500 ppm) for the next 26 weeks, then values were similar to those of the control group. For females of group 4, an increase in food intake was recorded during weeks 23 to termination. Higher ratios were obtained at week 1 for males and females of groups 4 and 5 (500 and 1500 ppm). Subsequent ratios for the males were similar to control ratios, whereas for the females, higher ratios were obtained from weeks 19 and 9 for groups 4 and 5, respectively. For the second year of the study, ratios for females of group 4 (500 ppm) became similar to those of the controls. Water intakes by males and females were not influenced by-treatment apart from transiently higher intakes recorded during weeks 16-28 for females of group 4 (500 ppm). Scheduled ophthalmological examinations gave no conclusive evidence of a treatment- related effect on the eyes. The week 13 examination prior to the sacrifice of group 5 (1500 ppm) revealed higher platelet counts in some males and higher white blood cell counts in some females. Subsequent examinations revealed no evidence of an influence on the haematological profile of treated rats. Lower plasma protein, plasma albumin and higher potassium and inorganic phosphorus values were recorded for females of group 5 (1500 ppm) at week 13. Treatment did not influence the blood chemistry profile of male rats or of females of groups 2, 3 and 4 (5, 50 and 500 ppm). Renal function, as assessed by urine analysis, was not disturbed by treatment. No treatment- related effects on organ weights were recorded. In the present study, treatment-related findings were observed only in groups 4 and 5 (500 and 1500 ppm) and did not indicate any oncogenic effect. Some lesions were found in both groups, and others only in group surviving the 2 year test period and not in group 5 which had to be sacrificed after 3 months of treatment due to clinical signs of toxicity. Macroscopic examination revealed higher incidences of mottled lungs in males and females of group 4. On microscopical examination, pulmonary alveolar foam cells were increased in incidence in males and females of groups 4 and 5 and additionally in severity in those of group 4. This lesion was considered associated with a dilatation of the right heart ventricle developing due to the increased pulmonary pressure caused by massive aggregations of foam cell in some females of group 4. In the non-glandular stomach ulcerative and inflammatory lesions were increased in incidence in males and females of group 4. In the cecum and/or in the colon, focal haemorrhagic, necrotic, ulcerative, and inflammatory lesions were found in males and females of groups 4 and 5. In the liver, increased incidence of fatty change of the peri lobular region was observed in female group 4. In addition, inflammation of the female urinary tract was markedly increased in incidence in group 4.

In conclusion, treatment with the test substance resulted in whole body tonic-clonic convulsions at 500 and 1500 ppm. The extent of the reaction showed that the maximum tolerable dose (MTD) was exceeded at the high dose level of 1500 ppm and this group was terminated in week 14. Dietary levels of 500 ppm initially resulted in a reduced body weight gain. In females this reversed to a marked increase in body weights from week 27 onwards, associated with a marked increase in food intake. Histopathological changes at 500 and/or 1500 ppm included an increase in the incidence of pulmonary alveolar foam cells, ulcerative and inflammatory lesions in the non-glandular stomach, and focal lesions in the caecum and/or colon. Additionally, increased incidences of fatty change in the liver and inflammation of the urinary tract were detected in females. There was no evidence of a tumorigenic response. It was concluded that the NOAEL was 50 ppm, corresponding to an average daily intake of 1.93 and 2.34 mg/kg bw/day in males and females respectively. The test substance showed no potential for carcinogenic effects in rats.