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

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 Nov 1997 - 9 Feb 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test procedure according to national standards

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

The study is also according to OECD guideline 474

GLP compliance:

yes

Type of assay:

micronucleus assay

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:

- Physical state: light-brown solid plaques
- Analytical purity: 98.1%
- Lot/batch No.: 232455681
- Stability under test conditions: yes

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Age at study initiation: 6 - 12 weeks
- Weight at study initiation: 38 - 42 g (male), 28 - 34 g (female)
- Assigned to test groups randomly: yes
- Housing: Individually in type I cages
- Diet: Altromin 1324 ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5 - 23
- Humidity (%): 45 - 50
- Air changes (per hr): ten times per hour
- Photoperiod (hrs dark / hrs light): 12 h light cycle

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5% aqueous Cremophor solution
- Amount of vehicle: 10 mL/ kg bw

Duration of treatment / exposure:

single application of test substance

Frequency of treatment:

once

Post exposure period:

16, 24 and 48 h

Doses / concentrations

Remarks:

Doses / Concentrations:
700 mg/kg body weight
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: i.p.
- Doses / concentrations: 20 mg/ kg bw

Examinations

Tissues and cell types examined:

erythrocytes in bone marrow (1000 cells)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Selection of the dose was based on a pilot study. Diuron was administered at concentrations of 500, 700, 1000 and 2000 mg/kg body weight to both sexes and animals were observed for 48 hours.
All animals treated with a dose of 2000 mg/ kg bw and 4 of 5 animals administered with a dose of 1000mg/kg bw died during the pretest.

DETAILS OF SLIDE PREPARATION: Schmidt`s method was used to produce the smear

METHOD OF ANALYSIS: A test was considered positive if, at any of the intervals, there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control. A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythocytes at any time. A test was also considered negative if there was a significant increase in that rate which, according to the laboratory`s experience was within the range of negative historical controls.

Evaluation criteria:

1000 cells were examined
Parameters:
- numbers and types of structural aberrations (micronuclei);
- polychromatic and normochromatic erythrocytes ratio

Statistics:

Groups were checked by Wilcoxon`s test and standard deviations were calculated

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Remarks:

no alterations reported

Toxicity:

yes

Remarks:

See any other informationon results incl. tables.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

- After single intraperitoneal administration of 700 mg/kg Diuron the following symptoms were observed for up to 48 hours: Apathy, roughened fur, staggering gait, sternal recumbency, spasm, twitching, difficulties in breathing, rapid breathing and eyelids closed.

Table 1: Summary of results of micronucleus test with Diuron (after acute intraperitoneal treatment with 700 mg/kg bw)

Experimental groups	Number of evaluated PE^1)	Number of NE^2)per 1000 PE^1)	Micronucleated cells per 1000
			NE^2)	PE^1)
Negative control	10,000	889±215	0.8±1.1	1.8±1.3
Diuron 16 hours	10,000	1200±246	0.7±0.7	2.6±1.8
Diuron 24 hours	10,000	1429*±592	0.7±0.8	2.2±0.9
Diuron 48 hours	10,000	1176±303	1.0±0.9	2.5±1.4
Positive control CPA 20 mg/kg	10,000	908±206	1.2±1.1	16.2**±6.4

* p< 0.05 in non-parametric Wilcoxon ranking test

** p< 0.01 in non-parametric Wilcoxon ranking test

1) polychromatic erythrocytes

2) normochromatic erythrocytes

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative

Executive summary:

A reliable micronucleus test in mice was conducted according to OECD 474 and EEC guidelines. After single i.p. treatment with 700 mg/kg bw Diuron no mortalities were observed, but all animals showed signs of toxicity. No increase in micronucleated PCEs over solvent control was noted at any sampling time point, so no indications of a clastogenic effect of Diuron were found. (Herbold, 1998).