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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Feb 1982 - Feb 1983
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report Date:
1985

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Analytical purity: 98.2 - 98.5 %
- Lot/batch No.: 232114156
- Stability under test conditions: yes

Test animals

Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Age at study initiation: 20 - 27 weeks
- Weight at study initiation: 6.2 - 9.9 kg
- Housing: dogs were kept in individual cages, (110 x 115 cm2 or 110 x 150 cm2)
- Diet: "ssniff HH sole diet for dogs, double ground" daily in the morning (week 1 to 5: 300 g, week 6 to 10: 330g, week 11 to 15: 350 g,
week 16 to 26: 380 g, week 27 to 40: 400 g, week 41 to 53: 430 g)
- Water: tap water ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 22
- Humidity (%): approx. 50
- Photoperiod (hrs dark / hrs light): 12 hour cycle

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: Ssniff HH sole diet for dogs
Details on oral exposure:
DIET PREPARATION
- The feed substance mixtures were produced in a mixer granulator type MGT.
- Mixing appropriate amounts with (Type of food): Ssniff HH sole diet for dogs
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Regulary analytical checks throughout the study ensured that the feed-substance mixtures actually contained the declared Diuron concentrations.
Before the start of the study it had been established that the test compound was stable for at least ten days in the dry feed and at least 24 hours in
wet feed, and was homogeneously mixed.
Duration of treatment / exposure:
12 months
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 300, 1800 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: substance concentrations administered were based on the results of a pilot test to establish dosage

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Behaviour and appearance were assessed several times daily
- mortality was checked several times daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- daily recorded as well as time of consumption

WATER CONSUMPTION: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to dosing, then at weeks 6, 13, 26, 39 and 52

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to dosing, then at weeks 6, 13, 26, 39 and 52
- How many animals: all
- Parameters checked: haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, MVV, MCH, MCHC, reticulocyte count, Heinz bodies, thromboplastin time and blood sedimentation rate.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to dosing, then at weeks 6, 13, 26, 39 and 52
- How many animals: all
- Parameters checked: sodium, potassium, chloride, calcium, glucose, urea, creatinine, total protein, ASAT, ALAT, alkaline phosphatise, GLDH,
bilirubin, cholesterol and serum proteins.

URINALYSIS: Yes
- Time schedule for collection of urine: performed on all animals at weeks 6, 13, 26, 39 and 52
- Parameters checked: pH, protein, glucose, occult blood, ketone bodies, bilirubin, specific gravity and sediment.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
-All organs were inspected grossly.
- Organ weights were determined for brain, heart, testes, adrenals, kidneys, liver, lungs, ovaries, pancreas, prostate, spleen, and thyroids.
HISTOPATHOLOGY: Yes
- Histopathology was performed for all animals on the following organs: adrenals, aorta, bone marrow, bone, brain, epididymes, oesophagus, eye, gall bladder, heart, kidney, liver, lung, lymph nodes, mammary gland, ovary, pancreas, pituitary gland, prostate, skeletal muscle, intestines, spleen, stomach, testis, thymus, thyroid gland, urinary bladder, uterus, parotis, nervi optici, nervi ischiacus.
Other examinations:
Body temperature and pulse rate were recorded in weeks -2, 6, 13, 26, 39 and 52.
Reflex tests were performed on each animal before start and in weeks 6, 13, 26, 39 and 52, including pupil reaction, corneal, patellar tendon,
stretch, righting and bending reflex.
Statistics:
Significance between controls and treated groups was analysed by Wilcoxon’s “two-sided test” (non-parametric rank sum).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
See details on result.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See detail on results.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See detail on results.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See detail on results.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See detail on results.
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
- No relevant adverse effects on behaviour or appearance for dogs of all groups were noted.
- Vomiting was observed in isolated cases and not related to treatment. Nature of faeces was not affected by Diuron-treatment.
- All animals survived the study.

BODY WEIGHT AND WEIGHT GAIN
- Body weight gain in dogs receiving 50 or 300 ppm was comparable to controls. At 1800 ppm a decrease in body weight gain was presenting the latter part of the study and more pronounced in females.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- Dosing at 50 and 300 produced no adverse effects on food consumption.
Some males of the top dose group left feed residues attributable to treatment.
Females in other groups did also not completely consume their diet, which is due to over-feeding, since feed ration was calculated over all animals.
- The mean quantities of Diuron taken up averaged for both sexes together were calculated to be
131.1 (group I), 778.3 (group II), and 4410.7 mg/animal and week (group III). When calculated for males on a weekly basis,
average uptake was 133.5, 794.1 and 4712.8 mg/animal/week for males in groups I, II and III, respectively.

OPHTHALMOSCOPIC EXAMINATION
- No ocular changes were noted in any of the treatment groups.

HAEMATOLOGY
- Thromboplastin times, blood sedimentation rates and differential blood counts were not affected by treatment.
Red blood cells were affected by Diuron as evidenced by reduced haemoglobin and erythrocyte count and increased mean corpuscular cell volumes of the red blood cells. Moreover, high numbers of Heinz bodies in erythrocytes and increasing reticulocytes in dogs dosed at 1800 ppm indicated an anaemic process and compensation reactions

CLINICAL CHEMISTRY
- Liver alterations were indicated by increased alkaline phosphatase in high dose animals. This finding was supported by slightly increased
cholesterol values. In top dose animals increased bilirubin values were observed pointing to a haemolytic icterus.

URINALYSIS
All results were comparable between controls and dosed dogs

ORGAN WEIGHTS
Testes in top dose males and liver and spleen in all animals of the 1800 ppm group were increased.

GROSS PATHOLOGY and HISTOPATHOLOGY: NON-NEOPLASTIC
Some histopathological findings were related to Diuron-treatment:
- Gross pathology showed dark colouration of bile, spleen, kidney, and bone marrow of mainly high dose animals.
- Iron-containing pigment deposits were found in the livers of top dose females in the Kupffer cells.
- Spleens of high and mid dose animals showed a high incidence of iron-containing pigments in the red pulp.
- Proximal tubuli or kidneys at the higher doses exhibited golden-brown crystalloid pigment.
- Reactive fat-deficient bone marrow with an increased iron.-content in animals of the 1800 ppm group was assessed.

OTHER FINDINGS
Examination of reflexes, pulse rates and body temperature did not reveal any treatment-induced alterations.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1.8 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: 50 ppm is equivalent to approximately 1.8 mg/kg/d
Dose descriptor:
LOAEL
Effect level:
11 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: 300 ppm is equivalent to approximately 11 mg/kg bw/d. Based on haemolytic anaemia, pigmentation of liver, kidneys and spleens and altered organ weights (liver, spleen)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Results of a 12 -month repeated dose toxicity study in dogs

Parameter

Control

50 ppm

300 ppm

1800 ppm

 

Male

Female

Male

Female

Male

Female

Male

Female

Observations

Number of animals examined

6

6

6

6

6

6

6

6

Mortality

0

0

0

0

0

0

0

0

Clinical signs

None (vomiting sporadically in all groups)

Body weight gain

NA*

NA

NA

NA

NA

NA

¿

¿

Food consumption

NA

NA

NA

NA

NA

NA

¿

¿

Mean compound intake [mg/kg bw/d]

0

0

1.8

11

64

Serum protein analysis

Alpha 2 globulin [%]

NA

NA

NA

NA

NA

NA

¿

¿

Beta globulin [%]

NA

NA

NA

NA

NA

NA

¿

¿

Organ weights

Liver

NA

NA

NA

NA

NA

NA

¿

¿

Spleen

NA

NA

NA

NA

NA

NA

¿

¿

Testes

NA

NA

NA

NA

NA

NA

¿

--

Histopathology [no. of incidence]

Livera)

0

0

1

1

1

0

5

6

Spleenb)

0

0

0

1

4

5

5

3

Kidneyc)

0

0

1

0

2

4

5

5

Bone marrowd)

0

0

0

0

0

1

5

3

NA – not affected

a)deposit of iron-containing pigment in Kupffer cells

b)iron-containing pigment in red pulp region

c)golden brown crystalloid pigment in proximal tubuli

d)reactive fat-deficient bone marrow with increased iron-content

Table 2: Results of clinical chemistry (group means of both sexes)

Dose group

Week

Alanine aminotransferase [U/L]

Alkaline phosphatase [U/L]

Bilirubin [µmol/L]

Chloesterol [mmol/L]

0 ppm

-2

6

13

26

39

52

21.11

20.03

24.83

21.12

27.72

23.62

259.1

203.8

198.1

176.6

189.6

147.7

2.62

3.42

4.07

3.35

3.42

4.66

3.084

2.981

3.014

3.279

3.815

3.125

50 ppm

-2

6

13

26

39

52

21.76

21.54

23.00

24.85

30.21

25.61

249.1

189.7

1889.3

158.1

142.2

121.2

2.82

3.18

3.52

2.87

3.17

3.86

2.942

2.975

2.909

3.414

3.774

2.911

300 ppm

-2

6

13

26

39

52

23.06

20.14

22.98

22.50

26.51

25.23

248.7

175.2

174.8

151.0

145.4

126.2

2.73

3.72

3.71

2.66

3.20

4.01

2.659

3.725

3.461

3.759

3.452

3.202

1800 ppm

-2

6

13

26

39

52

23.36

18.40

21.42

24.77

34.54

31.78

265.3

282.9**

368.2***

396.9***

552.5***

381.1***

3.12

4.82***

4.74

3.28

5.02***

5.16

2.981

4.348***

3.681

4.345***

4.372

3.589

* p =0.05      ** p =0.02    *** p =0.01

Table 3: Chronic toxicity of Diuron to dogs, summarized haematology data (group means from both sexes)

Treatment

Week

ERY.1)

1012/L

HB2)

g/L

 

HCT3)

L/L

MCV4)

fL

MCH5)

pg

MCHC6)

g/L

ERY

THROMB.7)

109/L

TPT8)

sec

LEUCO.9)

109/L

RETIC.10)

HEINZ.

BODIES

Control

- 2

6.203

139.3

0.4393

71.7

22.86

315.3

297.5

7.45

16.01

7.1

 

6

6.379

149.2

0.4482

69.3

23.8

331.9

238.5

7.78

11.63

4.7

 

13

6.374

148.4

0.4880

75.3

22.97

302.8

257.8

7.87

13.47

7.2

1.3

26

6.810

153.4

0.4865

71.7

22.57

318.1

264.4

7.21

13.44

4.6

0.5

39

6.741

158.7

0.4859

72.3

23.37

324.8

261.2

9.07

13.74

7.7

1.4

52

6.777

160.3

0.4558

67.9

23.70

350.3

237.8

8.99

13.76

10.4

2.8

 

 

 

 

 

 

 

 

 

 

 

 

 

Diuron

50 ppm

- 2

6.083

137.8

0.4397

71.8

22.73

312.3

288.8

7.50

16.02

7.8

 

6

6.118

142.8

0.4276

69.0

23.01

331.5

240.2

7.69

12.46

4.9

 

13

5.915

139.6

0.4555

75.8

23.26

306.3

265.6

7.91

13.70

5.4

1.4

26

6.677

148.9

0.4795

71.8

22.34

313.1

280.3

7.45

14.12

4.9

0.9

39

6.572

152.2

0.4748

72.6

22.98

318.7

262.0

8.64

13.00

5.2

1.3

52

6.745

160.2

0.4559

68.3

23.79

350.2

229.8

9.07

13.86

6.9

2.2

               

 

 

 

 

 

 

 

 

 

 

 

 

Diuron

300 ppm

- 2

6.229

141.0

0.4472

71.5

22.67

314.3

297.3

7.69

14.76

6.6

 

6

5.957*

139.6

0.4254

70.3

23.12

327.1*

298.3***

7.78

10.31

6.8

 

13

5.863**

139*

0.4627

77.4**

23.41

299.6

315.3***

7.87

11.99

8.0

3.7

26

6.477

144.9

0.4745

73.3**

22.41

307.7***

312.8**

7.47

13.67

6.3

1.9

39

6.270

150.3

0.4636

74.2*

23.72

322.0

323.8***

8.92

11.68

7.1

4.2

52

6.636

158.1

0.4551

69.2

23.80

345.9*

268.3

9.12

12.10

10.0

4.9

 

 

 

 

 

 

 

 

 

 

 

 

 

Diuron

1800 ppm

- 2

6.340

147.0

0.4608

72.5

23.22

317.3

295.6

7.64

16.08

7.0

 

6

5.490***

129.9**

0.4177

74.8***

23.32

309.9***

480.7***

7.65

13.67

40.9***

 

13

5.522***

132.5**

0.4550

80.8***

23.65

289.8***

455.7***

7.96

16.64

47.2***

204.2***

26

5.765***

132.3***

0.4417

76.5***

22.95

301.8***

419.8***

7.54

16.49*

44.3***

203.8***

39

5.663***

137.8***

0.4387**

77.5***

24.09

312.2***

425.0***

9.02

16.77

37.8***

409.2***

52

5.938***

142.6***

0.4257

72.3***

24.01

333.8***

359.4***

9.10

17.35***

38.3***

427.4***

1)erythrocyte counts    2)hemoglobin  3)hematocrit    4)mean corpuscular volume    5)mean corpuscular hemoglobin content   

6)mean corpuscular hemoglobin concentration 7)thrombocyte counts  8)thromboplastin time  9)leucocyte counts      

10)reticulocyte counts

* p =0,05      ** p =0,02    *** p =0,01

Applicant's summary and conclusion

Executive summary:

A reliable 12-month chronic toxicity study in dogs was conducted which was equivalent or similar to OECD 452 giudeline. Beagle dogs received 0, 50, 300 or 1800 ppm of Diuron, mixed into the standard dog diet. All animals survived and no treatment-related clinical signs were observed but effects on red blood cell parameters indicated a hypochromic haemolytic process from 300 ppm onwards.

The NOAEL is set to approx. 1.8 mg/kg/d (50 ppm) for both sexes and the LOAEL is assessed to be approx. 11 mg/kg bw/d (300 ppm), based on haemolytic anaemia, pigmentation of liver, kidneys and spleens and altered organ weights. (Hoffmann and Schilde, 1985)