diuron (ISO); 3-(3,4-dichlorophenyl)-1,1-dimethylurea

Repeated dose toxicity: oral

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

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Diuron

EC Number:

206-354-4

EC Name:

Diuron

Cas Number:

330-54-1

Molecular formula:

C9H10Cl2N2O

IUPAC Name:

3-(3,4-dichlorophenyl)-1,1-dimethylurea

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 or test system 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

1

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

2

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]
Liver^a)	0	0	1	1	1	0	5	6
Spleen^b)	0	0	0	1	4	5	5	3
Kidney^c)	0	0	1	0	2	4	5	5
Bone marrow^d)	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) 10^12/L	HB^2) g/L	HCT^3) L/L	MCV^4) fL	MCH^5^) pg	MCHC^6) g/L ERY	THROMB.^7) 10^9/L	TPT^8) sec	LEUCO.^9) 10^9/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)