Diquat dibromide

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 Dec 1985 to 10 Jan 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1983

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

2000

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Diquat dibromide

EC Number:

201-579-4

EC Name:

Diquat dibromide

Cas Number:

85-00-7

Molecular formula:

C12H12N2.2Br

IUPAC Name:

1,1'-ethylene 2,2'-bipyridyldiylium dibromide

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

liquid

Test animals

Species:

mouse

Strain:

C57BL

Remarks:

6J/Alp

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8 - 12 weeks (phase 1 - preliminary toxicity test), 10 - 12 weeks (phase 2 - micronucleus test)
- Weight at study initiation: 17.8 - 27.6 g (micronucleus test)
- Housing: Up to 10/cage (sexes separately) in stainless steel cages 32.5 cm x 18 cm x 12.5 cm
- Diet: Porton Combined Diet (PCD) ad libitum
- Water: filtered tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 23
- Humidity (%): 42 - 69
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 02 Dec 1985 To: 10 Jan 1986

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: deionised water
- Amount of vehicle: 10 mL/kg

Frequency of treatment:

Single dose

Post exposure period:

Bone marrow cells were harvested at 24, 48 or 72 hours post-treatment. An overview of the terminations times for the different treatment and control groups can be found in Table 1 in 'Any other information on materials and methods incl. tables'.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

62.5 other: mg/kg bw/day based on test substance cation species

Remarks:

Lower dose group

Doses / concentrations 2

Dose / conc.:

100 other: mg/kg bw/day based on test substance cation species

Remarks:

Higher dose group

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Concentration in vehicle: 65 mg/kg
- Route of administration: single oral administration

Examinations

Tissues and cell types examined:

Bone marrow Polychromatic erythrocytes (PCEs)

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION
All animals designated for bone marrow smears were killed by cervical dislocation at 24, 48 or 72 hours after receiving a single dose of the test substance. Femurs were removed and stripped clean of muscle. The iliac end of the femur was removed and a fine paint brush was wetted with a solution of albumin (6% w/v in physiological saline). This was then dipped into the marrow canal and four smears were painted on an appropriately labelled clean, dry microscope slide. The slides were allowed to air dry and were stained with polychrome methylene blue and eosin using an automatic staining machine.

CRITERIA FOR DOSE SELECTION
Preliminary toxicity testing

METHOD OF ANALYSIS
Slides were coded and scored blind. One thousand polychromatic erythrocytes were examined and the number containing micronuclei scored. The slides were also examined for evidence of cytotoxicity, which may be manifest by alterations in the ratio of different cell types in the bone marrow. This was assessed by counting the ratio of polychromatic to normochromatic erythrocytes in a sample of 500 erythrocytes.

Statistics:

Analysed for significant difference from the vehicle control group using a one-sided Student’s ‘t’ test.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING STUDY
Initially diquat was dosed at 50, 150 and 250 mg/kg. Due to the mortality observed additional dose levels of 100 and 128 mg/kg were tested. The mortalities were: 0, 0, 1, 2 and 4 for males and 0, 0, 5, 3 and 5 for females at dose levels of 50, 100, 128, 150 and 250 mg/kg diquat respectively. The dose levels used in the main micronucleus test were 80 % and 50 % of the MLD (i.e. 100 and 62.5 mg/kg).

MICRONUCLEUS TEST RESULTS
No biologically or statistically significant increase in mean frequency of polychromatic erythrocytes (PCEs) containing micronuclei was seen at any dose level at any of the sampling times. A biologically and statistically significant reduction in number of PCEs to normochromatic erythrocytes (NCEs) was observed with both dose levels of diquat at all 3 sampling times with the exception of 62.5 mg/kg at 24 hours, indicating a cytotoxic effect. The test system positive control, cyclophosphamide, induced statistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen.

Any other information on results incl. tables

Table 2: Mean incidence of micronuclei/1000 polychromatic erythrocytes

Treatment	Dose	Time of kill
		24 hours	48 hours	72 hours
Vehicle control (deionised water)	10 mL/kg	1.7	2.0	1.6
Cyclophosphamide (positive control)	65 mg/kg	19.3**	14.7**	3.4*
Test substance	62.5 mg/kg	1.7 (9)	2.3	1.2
	100 mg/kg	2.5	1.3	2.5
all mean values based on 10 observations except where indicated in parentheses * statistically significantly different at p < 0.05 ** statistically significantly different at p < 0.01

Applicant's summary and conclusion

Conclusions:

There were no significant increases in micronucleated polychromatic erythrocytes at doses of 62.5 or 100 mg test substance/kg bw. The test substance is not clastogenic in the mouse micronucleus test.

Executive summary:

In a bone marrow micronucleus assay with C57BL/6J/Alpk mice, performed according to OECD TG 474 under GLP, 5 mice/sex/group were given a single oral dose of test substance at doses of 100 and 62.5 mg test substance cation/kg, equivalent to 80% or 50% of the median lethal dose. Bone marrow cells were harvested at 24, 48 or 72 hours post-treatment, smears were prepared and stained with polychrome methylene blue and eosin. Cells were scored for the incidence of micronucleated polychromatic erythrocytes, and also for the percentage of polychromatic erythrocytes in the erythrocyte population. The vehicle was deionised water and the positive control was cyclophosphamide.There were no signs of toxicity during the study. The test substance did not induce a biologically or statistically significant increase in polychromatic erythrocytes containing micronuclei, when tested at dose levels of 00 and 62.5 mg test substance cation/kg corresponding to 186.8 and 116.75 mg/kg pure test substance and equivalent to 80% and 50% of the median lethal dose (MLD) in C57BL/6J/Alpk mice. The positive control induced the appropriate response. Test substance was not clastogenic in the mouse micronucleus test.