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Toxicological information

Genetic toxicity: in vivo

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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:
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

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
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 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 PE1)

Number of NE2)per 1000 PE1)

Micronucleated cells per 1000

NE2)

PE1)

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).