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Toxicological information

Carcinogenicity

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Description of key information

- The NOAEL for systemtic toxicity was established at 50 ppm (corresponding to an average daily intake of 1.93 and 2.34 mg/kg bw/day in males and females, respectively) and the NOAEL for carcinogenicity is set >1500 ppm (corresponding to an average daily intake of  108 and 114 mg/kg bw/day for males and females, respectively), rat, chronic, dietary, OECD TG 453, Bachmann 1993.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Referenceopen allclose all

Endpoint:
carcinogenicity: oral
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:
1981
Qualifier:
according to guideline
Guideline:
EPA OPP 83-5 (Combined Chronic Toxicity / Carcinogenicity)
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)
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
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on 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
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
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

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.
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.
Dermal irritation (if dermal study):
not examined
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
Key result
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
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: Dietary equivalent to 1.93 and 2.34 mg/kg bw/day for males and females, respectively.
Key result
Dose descriptor:
NOAEL
Remarks:
Carcinogenicity
Effect level:
> 1 500 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Dietary equivalent to 108 and 114 mg/kg bw/day for males and females, respectively.
Key result
Critical effects observed:
no

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

* 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].

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 and therefore the NOAEL is set >1500 ppm (corresponding to an average daily intake of 108 and 114 mg/kg bw/day in males and females, respectively).
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 and therefore the NOAEL is set > 1500 ppm (corresponding to an average daily intake of 108 and 114 mg/kg bw/day in males and females, respectively).

Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 Jan 1990 to 27 Aug 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 451 (Carcinogenicity Studies)
Version / remarks:
1981
Qualifier:
according to guideline
Guideline:
EPA OPP 83-2 (Carcinogenicity)
Version / remarks:
1982
Qualifier:
according to guideline
Guideline:
other: Guidance on Toxicology Study data for Application of Agricultural Chemical Registration, 59 Noh San No. 4200
Version / remarks:
1985
GLP compliance:
yes (incl. QA statement)
Species:
mouse
Strain:
other: Tif: MAGf (SPF), hybrids of inbred MAG x NIH
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: Approx. 4 weeks
- Weight at study initiation: 25.61 - 30.90 g in males and 22.02 - 27.19 g in females
- Housing: Male animals were housed individually in macrolon cages type 2, and the females in groups of 5 animals in macrolon cages type 3, both with wire mesh tops and sterilised, granulated soft wood bedding
- Diet: Pelleted, certified 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: 12 Jan 1990 to 27 Aug 1991
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet: Fresh diets were prepared every four weeks throughout the study.
- Mixing appropriate amounts with type of feed: The test substance was weighed on a calibrated balance. The pulverised food was then homogeneously mixed with the appropriate concentrations of the test article and about water was added before pelleting to ensure the necessary pellet quality. The pellets were subsequently airdried and stored in stainless steel containers in a separate area, 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:
18 months
Frequency of treatment:
Continuously
Dose / conc.:
2 ppm
Remarks:
Group 2: Mean dietary equivalent to 0.222 and 0.217 mg/kg bw/day for males and females, respectively
Dose / conc.:
20 ppm
Remarks:
Group 3: Mean dietary equivalent to 2.25 and 2.12 mg/kg bw/day for males and females, respectively
Dose / conc.:
200 ppm
Remarks:
Group 4: Mean dietary equivalent to 22.6 and 22.0 mg/kg bw/day for males and females, respectively
Dose / conc.:
400 ppm
Remarks:
Group 5: Mean dietary equivalent to 62.9 and 61.2 mg/kg bw/day for males and females, respectively
No. of animals per sex per dose:
60 animals per sex per dose were used, consisting of the following Experimental groups:
Experimental group I: 50 animals per sex per dose for evaluation of carcinogenic potential
Experimental group II: 10 animals per sex per dose for evaluation of carcinogenic potential and of haematological parameters
Control animals:
yes, plain diet
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: Daily, records at least weekly for in life observations and daily (a.m. and p.m. on working days, a.m. on weekends and holidays) for 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.

HAEMATOLOGY:
- Time schedule for collection of blood: Week 53 and 78.
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: For terminal laboratory investigations at week 78, the number of animals subjected to examination was fortified by animals of the carcinogenicity group to yield 10 samples/sex/group where possible.
- Parameters checked: In week 53: erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, leukocyte count, leukocyte differential count, and thrombocyte count
- Parameters checked: In week 78 where a complete blood cell counts and white blood cell differential counts based on light scattering and peroxidase cytochemistry: Erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, Red cell volume distribution width, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, mean leukocyte count, mean leukocyte differential count (neutrophils, eosinophils, basophils, lymphocytes, lymphocytes, monocytes, large unstained cells), blood platelets and thrombocyte count.
Sacrifice and pathology:
GROSS PATHOLOGY
MACROSCOPICAL EXAMINATION:
- At the end of the test period the surviving controls and treated animals were bled under ether anaesthesia and subjected to detailed autopsy. At autopsy the following weights were recorded from all animals: body (exsanguinated), brain, liver, kidneys, adrenals, gonads (testes/ovaries).
- The following organs and tissues 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, gall bladder, pancreas, oesophagus, 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, extra-orbital lacrimal gland, both, Zymbal gland, both, muzzle, tongue and any tissue with gross lesions.
- Unless prevented by advanced autolysis, cannibalism or by technical reasons a complete autopsy with tissue preservation was performed also on all animals which died during the test period or which had to be sacrificed in condition. No or organ weights were recorded for these animals. Because of signs of toxicity with high mortality, all surviving animals of group 5 (400 ppm) were sacrificed in weeks 9 and 10 and this treatment group did not take part in the evaluation of the chronic study. These animals were also subjected to a detailed autopsy including tissue preservation; carcass or organ weights were not recorded.

MICROSCOPICAL EXAMINATION:
- After the fixation, organ samples listed below were taken, embedded in paraplast, sectioned at 3 - 5 microns, stained with haematoxylin and eosin, and subjected to microscopical examination: skin, mammary area, spleen, mesenteric lymph node, axillary lymph node, sternum with bone marrow, femur with joint, skeletal muscle, trachea, lung, heart, aorta, submandibular salivary gland, both, liver, gall bladder, pancreas, oesophagus, 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, extra-orbital lacrimal gland, both, any tissue with gross lesions.
- Specific stains were utilised if necessary: Sudan in selected liver specimens to characterise hepatocellular vacuoles; furthermore Van Gieson, periodic acid-Schiff-reaction, methenamine-silver, acid fuchsin-orange G and Pearl's iron in selected tissues. Immunocytochemical stains (LU-5 and S-100) were performed in selected cases to determine the tissue of origin for particular tumours.
- Animals of group 5 (400 ppm), for reasons mentioned before, were not taken into consideration for establishing a possible treatment-related effect by microscopy in comparison with the other treatment groups. Nevertheless, microscopical findings with higher incidences than those normally observed in animals of our colony of mice used in three-months studies were reported.
- Diagnostic criteria used for the description of lesions were based on the publications listed at the end of this section. All organs from ten percent of randomly selected animals of both sexes, all target organs and all unusual lesions were revaluated by the reviewing pathologist.
- Analysis and evaluation of pathology data: To establish a possible relationship to the treatment a statistical analysis of all microscopical findings occurring in at least one animal was performed. Where appropriate, lesions were combined and subjected again to statistical analysis for determining a possible effect of the treatment. In the presence of statistical evidence of a treatment effect its toxicological relevance was evaluated considering biological factors such as (1) the nature of the lesion, (2) number of affected animals, (3) whether the effect was dose-related, (4) whether the increase was associated with an increase in related lesions, (5) the historical control rate of the lesion in question, (6) whether the effect was observed in a suspected target organ, (7) the relative survival of treated and control animals, and (8) the appropriateness of combining lesions of varying sites and histogenesis.
Microscopical findings without statistical evidence of a treatment effect were reviewed taking consideration the biological factors mentioned above in order to detect further relevant changes.
Statistics:
For each time point and parameter an univariate statistical analysis was performed. Nonparametric methods <1> 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 <2> and tested for increasing or decreasing trends from control up to the respective dose group by Jonckheere’s test for ordered alternatives <3>. 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. Statistical tests and flags used are indicated in the header of each table. Survival analysis was performed by the regression model (partial likelihood) introduced by Cox <4> 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.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Tonic-clonic convulsions, occurring spontaneously or in response to external stimuli (e.g. handling), were exhibited by a number of males and females in groups 4 and 5 (200 and 400 ppm). The convulsions were of short duration (up to 30 seconds) during which time forelimb paddling, uncoordinated movement, jumping and straub tail occurred. A period of inactivity generally followed before normal behaviour resumed. within this colony of mice, a low frequency of spontaneous convulsive episodes has previously been recorded in untreated mice. In the present study, four control males were seen to convulse, in one of which five convulsive episodes during a 13 week period were observed. The convulsions recorded in controls and in groups 2 and 3 (2 and 20 ppm) were of a shorter duration and less intense (tonic phase only) than those recorded in groups 4 and 5 (200 and 400 ppm) and in females the incidences showed no dose-relationship. Therefore, the convulsive episodes recorded animals of groups 2 and 3 (2 and 20 ppm) are regarded as spontaneous events, unrelated to treatment.
No other signs of reaction to treatment were observed. In particular, the incidences of superficial masses were similar in treated and control groups
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
In group 5 (400 ppm), during the first 9 weeks of the study, 5 males and 29 females were found dead. Therefore, all survivors of this group were sacrificed in weeks 9/10. A higher mortality was recorded for males and females of group 4 (200 ppm). The mortality recorded in groups 2 and 3 (2 and 20 ppm) was similar to that in the control group. Further mortality statistics (survival estimate) were performed as part of the statistical analysis of pathology data. The result showed a significant increase in mortality among group 4 animals (200 ppm).
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weight gains for treated males were slightly higher than control gains during the first few weeks of the study. Thereafter, mean values were similar to those of the control group. Body weight stasis was apparent for females of group 5 (400 during weeks 6 to 9, at which point the group was removed from the study. Mean body weights for females of groups 2, 3 and 4 (2, 20 and 200 ppm) remained similar to control values during the first year of treatment. Thereafter, slightly lower mean values were recorded for females of group 4 (200 ppm). Lower values were also recorded for females of group 2 (2 ppm) after one year although the absence of an effect on group 3 bod yweights suggest the difference for group 2 was not treatment-related.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
During the nine weeks of treatment the food intakes for males of group 5 (400 ppm) were similar to control values. For females of group 5 the overall food intake during this period was lower than that of the controls. During the first 9 weeks of the study, males of groups 2, 3 and 4 (2, 20 and 200 ppm) showed a tendency towards minimally higher food intakes (4 % to 6 % higher) compared with the controls. However, these increased values were considered associated with a group disparity in the pre-treatment week, where the intakes were already 3 – 5 % above the control. Therefore, a relation to treatment can be excluded. The food intakes for females of groups 2, 3 and 4 (2, 20 and 200 ppm) were similar to control values throughout the treatment period.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
Calculated food consumption ratios revealed slightly lower values for females of group 5 (400 ppm) whereas the ratios for males of this group were similar to those of the controls. The ratios obtained for males and females of groups 2, 3 and 4 (2, 20 and 200 ppm) were essentially similar to those of the control group throughout the study.
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):
Treatment had no effect on the haematological profile of the mice. At the 78 week investigation, the mean white blood cell count for group 3 (20 ppm) was unduly influenced by the high counts recorded for male 170 and female 479. Lymphatic leukaemia was diagnosed for these animals. However, no toxicological relevance is attached to these isolated findings of a pathology which occurs spontaneously in this colony of mice.
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
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):
The mean adrenal weight and ratio for females of group 4 (200 ppm) were higher than control values. However, all values were within the range recorded among controls and no morphological changes were detected by microscopy. other organ weights and ratios were not influenced by treatment.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The macroscopical examination of the animals of both sexes of group 5 (400 ppm), which all died spontaneously or were sacrificed in 9 and 10, revealed no treatment-related findings.
Lung: In male animals, nodules of the lung occurred in higher numbers in group 2 (2 ppm) and group 4 (200 ppm) whereas in animals of group 3 (20 ppm) the incidence was similar to that in control animals. Also an increase in multiple occurrence of nodules was observed in males of Group 4 (200 ppm). Masses of the lung were not increased in incidence in a treatment-related manner. All other findings occurred in comparable numbers in experimental groups 1 to 4 and were similar to those occurring spontaneously in our colony of mice. Thus, no experimental relevance is attributed to these findings.

Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Liver: Animals of both sexes of group 4 (200 ppm) presented with significantly higher numbers of fatty change in the liver. The degree of this hepatic lipidosis was predominantly moderate to marked. The lipidosis presented morphologically as a micro vesicular and/or macro vesicular type and the distribution within the hepatic lobule was diffuse, periportal or centrilobular. Among mice of group 5 (400 ppm), which all died spontaneously or were sacrificed in weeks 9 and 10, 37/60 males and 52/60 females were affected with fatty change in the liver. These incidences are higher than those observed in animals of our strain of mice used in three-months studies. In females, the number of necrotic changes in the liver was significantly increased in group 4 (200 ppm) compared with the control animals. In the majority of cases, these lesions presented as focal or multifocal hepatocellular necrosis or recent necrosis of small groups of hepatocytes, often in the neighbourhood of degenerated hepatocytes with fatty change. The degree of liver necrosis was generally slight. Females of group 5 (400 ppm) also presented with increased numbers (26/60) of necrosis in the liver when compared with control mice of our strain used in three-months studies.
- Prostate: Males of group 4 (200 ppm) presented with significantly higher numbers of inflammatory lesions of the prostate, in particular chronic inflammation. Furthermore, the occurrence of cystic dilatation of the prostatic glands was significantly increased in this treatment group. However, the degree of these degenerative changes was slight.
- Spleen: The occurrence of hemosiderosis of generally slight to moderate degree was significantly higher in animals of group 4 (200 ppm) in both sexes. However, when the incidences of animals sacrificed at the termination of the study were analysed, no treatment-related increase of hemosiderosis was observed in both sexes. Furthermore, no indication of an increased degradation of blood corpuscles was observed at the laboratory investigation. Therefore, this finding is considered as related to the mode of death, which may be preceded by a period of venous congestion inducing hemosiderosis in debilitated animals, rather than as a sign of underlying toxicity. 30/60 female animals of group 5 (400 ppm), which died or were sacrificed in moribund condition, also presented with increased numbers of this lesion compared with mice of our strain used in three-months studies.
- Lung: Females in group 4 (200 ppm) showed significantly higher numbers (18/60 versus 0/60 in controls) of congestion of the lung. However, no treatment-related cardiac effect was observed. This finding was present only in animals which died spontaneously and is therefore considered to be related to the mode of death of these animals rather than indicative of underlying toxicity. The same finding was observed in 26/60 females of group 5 (400 ppm) which were found dead.
- Kidney: Lymphohistiocytic infiltration occurred in 5/60 females of group 4 (200 ppm). Compared with the control animals (5/60), this finding was significantly increased when considering the higher mortality of group 4. No treatment-related effect was observed for this lesion, when the various inflammatory lesions of the kidney listed in the summary tables, including inflammation with fibrosis, inflammatory cell infiltration, lymphocytic infiltration and lymphohistiocytic infiltration were combined (7/60 versus 12/60 in controls). These lesions are considered for the majority of cases to be signs of beginning chronic progressive nephropathy in mice which, however, did not occur in a treatment-related manner. Therefore, this finding is considered to be incidental.
- Thyroid gland: The occurrence of chronic necrotising inflammation in the thyroid gland was significantly increased in group 4 (200 ppm) in females (2/60 versus 0/60 in controls). Because of the low number of affected animals, this finding is considered to be incidental. Additionally, a variety of other changes was found in this study. They commonly occur in our colony of mice, and, neither their incidences nor their distribution and morphologic appearance gave any indication of a treatment-related association. Furthermore, there were no histological findings indicating the cause of higher mortality in both sexes of groups 4 (200 ppm) and 5 (400 ppm).
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Proliferative lesions: The following tumours occurred in higher numbers in treated animals: Male animals bearing adenomas presented with significantly higher numbers in group 4 (200 ppm) on performing a survival-adjusted statistical analysis (p < 0,01). Furthermore, the absolute numbers of adenomas per treatment group (multiplicity analysis) were also significantly increased in group 4 (p < 0,001). The macroscopically increased incidences of single and multiple nodules of the lung in group 4, appeared to correlate with the adenomas diagnosed microscopically. In interpreting this result it must be considered that the true incidence of this frequent tumour in mice is much dependent on the sampling technique. Routine sectioning may not be appropriate to reveal the distribution of multiple tumours. For instance, serial sectioning of the lung increased the incidence of pulmonary adenomas and/or carcinomas from 30 % to 50 %. In the present study a sampling error is less likely however, since an increase in the number of smaller sized adenomas was demonstrated in the routine sections to have occurred in group 4 males.
The overall increase of single and multiple neoplastic lesions in group 4 was solely due to the higher occurrence of adenomas in the lung without an increase of the occurrence of carcinomas. The incidences of adenoma in the lung (from 6.5 % to 31.5 % showed no dose-related increase. The onset of lung tumours was late in the study (median day of diagnosis later than 540), with the majority of tumours detected at the terminal sacrifice. Therefore, the neoplastic lesions of the lung did not affect the survival, as illustrated by the stem and leaf plot in the statistical section of this report. There is to be noted, that the experimental incidence of lung adenomas and carcinomas is known to be very variable in aged mice. Depending on the mouse strain, spontaneous incidence rates of up to 90 % have been reported to occur in 12 - 18 months old mice.
The comparison with our historical control data showed that the numbers of lung adenomas in this study were comparable to the historical data for studies of at least two years, although only one study presented the same high incidence of lung adenomas as group 4 (200 ppm) of the present study. Because mice present usually a high increase in the occurrence of spontaneous lung tumours between 18 and 24 months of life, a comparison with such historical control data remains doubtful. Considering therefore the available historical data for studies of 18 months duration, as is the present study, animals of group 4 (200 ppm) presented distinctly more single (31.5 %) and multiple (46.5%) adenomas than the historical controls (15 % and 19 %) respectively) of our strain of mice. On the other hand, the incidence observed for pulmonary adenomas in control males of the present study (20 %) was already above the upper limit of the historical control data for 18 months old mice mentioned above.
In conclusion, the experimental relevance of the lung adenomas in male mice in group 4 (200 ppm) is considered as equivocal because of the following reasons: The incidence of both single and multiple adenomas of the lung, correlating with macroscopically reported increased numbers of lung nodules was slightly, but statistically significantly higher (p < 0,01 and p < 0,001 respectively) than in the controls and was distinctly above (31.5%) the upper limit of the historical control data for 18 months old males (15 %) of our strain of mice. However, the incidence of pulmonary adenomas observed in male control mice of the present study (20 %) was already beyond the upper limit of the historical controls of 18 months old males (15 %) and the incidences observed in the treated groups (from 6.5 % to 31.5 %) were not related to the doses applied. In addition, the incidences of lung adenomas are known to be very variable in aged mice and depending on the mouse strain, spontaneous incidence rates of up to 90 % have been reported.
The single male animal in the control group presenting macroscopically with multiple nodules revealed at microscopical examination a lipoma and an adenoma of the lung. Therefore, it was not counted as an animal bearing multiple epithelial (i.e. adenoma or carcinoma) tumours of the lung. Female mice did not present any treatment-related effect on neoplastic lesions of the lung. The analysis of the general occurrence of primary tumours in both sexes gave no indication of a treatment-related effect in early deaths or in animals sacrificed at the termination of the study.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Effect level:
20 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Remarks on result:
other: Mean dietary equivalent to 2.25 an 2.12 mg/kg bw/day for males and females, respectively
Key result
Dose descriptor:
NOAEL
Remarks:
Carcinogenicity
Effect level:
400 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Mean dietary equivalent to 62.9 an 61.2 mg/kg bw/day for males and females, respectively
Key result
Critical effects observed:
no
Conclusions:
The no observed adverse effect level was 20 ppm, equivalent to an average daily intake of 2.25 or 2.12 mg/kg body weight for males and females, respectively. The test substance did not present carcinogenic properties in mice.
Executive summary:

In the 18-month Tif: MAGf (SPF), hybrids of inbred MAG x NIH mouse study, animals were fed diets containing the test substance at 0, 2, 20, 200 or 400 ppm, equivalent to 0, 0.22, 2.25, 22.6, 62.9 mg/kg bw/day for males and 0, 0.22, 2.12, 22, 61.2 mg/kg bw/day for females according to OECD TG 451 and GLP principles. A total of 600 mice, 60 males and 60 females per dose group were used in this study. The food samples were analysed for concentration, homogeneity and stability. In life observations, mortality, body weight, food consumption (ratios), test substance intakes, haematology, macroscopic and microscopic examinations were analysed.

Results showed that dietary levels of 200 and 400 ppm lufenuron exceeded the maximum tolerated level for mice. Treatment at 400 ppm resulted in excessive mortality and this group was therefore terminated early. At 200 ppm the survival of both sexes was significantly reduced over the 18-month period. At both the 200 and 400 ppm level, tonic-clonic convulsive episodes occurred either spontaneously or in response to external stimuli. Treatment at 200 ppm resulted in higher incidences of fatty liver, which, in females, was accompanied by necrotic changes. Similar liver pathology was also observed in the 400 ppm group. In addition, males in the 200 ppm group had a higher incidence of inflammatory changes in the prostate. Males in the 200 ppm group showed an increased incidence of both single and multiple lung adenomas. The incidences observed across the treated groups in this study were not dose related, indicating that the variation in incidence was spontaneous rather than as a result of treatment.

In conclusion, the no observed adverse effect level was 20 ppm, equivalent to an average daily intake of 2.25 or 2.12 mg/kg body weight for males and females, respectively. The test substance did not present carcinogenic properties in mice.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
108 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
GLP compliant OECD TG 453 study

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data classification for carcinogenicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.

Additional information

All available data was assessed and the studies representing the worst-case effects was included as key study. The test substance showed no carcinogenic effects in rats (Bachmann, 1993) and in mice (Bachmann, 1993).

Oral: combined carcinogenicity study, 24-month dietary study in rats, Bachmann 1993

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 ratsand therefore the NOAEL is set > 1500 ppm (corresponding to an average daily intake of 108 and 114 mg/kg bw/day in males and females, respectively).

Oral: combined carcinogenicity study, 18-month dietary study in mice, Bachmann 1993

In the 18-month Tif: MAGf (SPF), hybrids of inbred MAG x NIH mouse study, animals were fed diets containing the test substance at 0, 2, 20, 200 or 400 ppm, equivalent to 0, 0.22, 2.25, 22.6, 62.9 mg/kg bw/day for males and 0, 0.22, 2.12, 22, 61.2 mg/kg bw/day for females according to OECD TG 451 and GLP principles. A total of 600 mice, 60 males and 60 females per dose group were used in this study. The food samples were analysed for concentration, homogeneity and stability. In life observations, mortality, body weight, food consumption (ratios), test substance intakes, haematology, macroscopic and microscopic examinations were analysed.

Results showed that dietary levels of 200 and 400 ppm lufenuron exceeded the maximum tolerated level for mice. Treatment at 400 ppm resulted in excessive mortality and this group was therefore terminated early. At 200 ppm the survival of both sexes was significantly reduced over the 18-month period. At both the 200 and 400 ppm level, tonic-clonic convulsive episodes occurred either spontaneously or in response to external stimuli. Treatment at 200 ppm resulted in higher incidences of fatty liver, which, in females, was accompanied by necrotic changes. Similar liver pathology was also observed in the 400 ppm group. In addition, males in the 200 ppm group had a higher incidence of inflammatory changes in the prostate. Males in the 200 ppm group showed an increased incidence of both single and multiple lung adenomas. The incidences observed across the treated groups in this study were not dose related, indicating that the variation in incidence was spontaneous rather than as a result of treatment.

In conclusion, the no observed adverse effect level was 20 ppm, equivalent to an average daily intake of 2.25 or 2.12 mg/kg body weight for males and females, respectively. The test substance did not present carcinogenic properties in mice.