Dimethylammonium 2,4-dichlorophenoxyacetate

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: EPA-Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation, Human and Domestic Animals, Series 81, 82, and 83, US Department of Commerce, National Technical Information Service PB91-154617, 1991.

Deviations:

not specified

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Dimethylammonium 2,4-dichlorophenoxyacetate

EC Number:

217-915-8

EC Name:

Dimethylammonium 2,4-dichlorophenoxyacetate

Cas Number:

2008-39-1

Molecular formula:

C8H6Cl2O3.C2H7N

IUPAC Name:

dimethylammonium 2,4-dichlorophenoxyacetate

Details on test material:

- Name of test material (as cited in study report): 2,4-D DMA, 2,4-D dimethylamine salt
- Analytical purity: 66.2% active ingredient
- Source of the test material: Industrial Task Force II on 2,4-D Research Data

Test animals

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague-Dawley, Frederick, MD, USA
- Diet: commercial diet "Purina certified Laboratory Chow (R) #5002” ad libitum
- Water: ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approx. 21
- Humidity (%): approx. 50
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

sterile deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
2,4-D DMA was mixed with sterile deionized water

Duration of treatment / exposure:

single administration of 10 mL/kg body weight of the dosing solution

Frequency of treatment:

single application

Post exposure period:

24, 48 and 72 h

Doses / concentrations

Remarks:

Doses / Concentrations:
39.7, 132, 397 mg/kg based on the active ingredient
Basis:
nominal in water

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral (gavage)
- Doses / concentrations: 80 mg/kg

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Doses were selected based on mortality results in range finding studies (3 mice/group).
Dose levels of 2,4-D DMA were as follows: 265, 530, 794, 1060 and 1324 mg/kg.
A top dose of 397 mg/kg was chosen based on mortality at doses of 530 mg/kg and above.

DETAILS OF SLIDE PREPARATION:
Bone marrow samples were collected at 3 intervals after treatment by removing the adhering soft tissue and epiphyses of both femurs,
and flushing or aspirating the marrow into a centrifuge tube utilizing a volume of foetal calf serum. After centrifugation to pellet the tissue, most of the supernatant was drawn off, the cells were resuspended, and the suspension spread on slides and air-dried.
The slides were then fixed, stained in either May-Gruenwald/Giemsa or Wright-Giemsa.

Evaluation criteria:

1000 polychromatic erythrocytes (PCE) were evaluated from each animal for the incidence of micronucleated polychromatic erythrocytes (MN-PCE).
Treatment induced perturbation in bone marrow erythropoiesis was ascertained by determining the relative proportion of PCE to normochromatic
erythrocytes (NCE).

Statistics:

Analysis of the data was performed using an analysis of variance on the square root arcsine transformation which was performed on the proportion of cells with micronuclei per animals followed by Tukey`s Studentized Range test (HSD) with adjustments for multiple comparison.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Table 1:Pattern of mortality from range-finding studies of 2,4-D DMA

Dose (mg/kg body weight)	Sex	No. of dead animals
265	Male	0/3
	Female	0/3
530	Male	1/3
	Female	0/3
794	Male	3/3
	Female	2/3
1060	Male	3/3
	Female	3/3
1324	Male	3/3
	Female	3/3

Table 2: Results of the in vivo micronucleus test

Treatment (mg/kg)^a	Sex	24 h sacrifice		48 h sacrifice		72 h sacrifice
		% MN-PCE^b	Ratio PCE/ NCE^c	% MN-PCE	Ratio PCE/ NCE	% MN-PCE	Ratio PCE/ NCE
Negative control 10 mL/ kg water	Male	0.06 ± 0.04^d	0.45 ± 0.07	-	-	-	-
	Female	0.06 ± 0.06	0.84 ± 0.07	-	-	-	-
Positive control 80 mg/ kg cyclophosphamide	Male	1.48 ± 0.35*	0.57 ± 0.06	-	-	-	-
	Female	2.12 ± 0.36*	0.87 ± 0.15	-	-	-	-
39.7	Male	0.04 ± 0.02	0.51 ± 0.03	0.10 ± 0.03	0.72 ± 0.05	0.12 ± 0.07	0.57 ± 0.07
	Female	0.02 ± 0.02	0.77 ± 0.08	0.06 ± 0.06	0.73 ± 0.17	0.08 ± 0.04	0.75 ± 0.13
132	Male	0.06 ± 0.04	0.47 ± 0.08	0.12 ± 0.05	0.62 ± 0.12	0.06 ± 0.06	0.79 ± 0.17
	Female	0.08 ± 0.06	0.08 ± 0.13	0	0.66 ± 0.12	0.04 ± 0.04	0.70 ± 0.08
397	Male	0.12 ± 0.06	0.33 ± 0.05	0.02 ± 0.02	0.55 ± 0.06	0.18 ± 0.06	0.48 ± 0.03
	Female	0.04 ± 0.02	0.75 ± 0.10	0.06 ± 0.04	0.47 ± 0.09	0.08 ± 0.04	0.38 ± 0.13

There was no significant increase in the frequency of micronucleated polychromatic erythrocytes.

 

- = not done

* = significantly greater than the corresponding vehicle control, p < 0.05

a = based on active ingredient content

b = Percent micronucleated cells based on the total polychromatic cells present in the scored optic field.

c = Ratio of polychromatic (PCE) to normochromatic cells (NCE), based upon the number of NCE in the optical fields containing 1000 PCE.

d = data are means and standard deviations

No increase in the incidence of micro-nucleated polychromatic erythrocytes in any sex or at any time point in the treated mice was observed. Therefore no clastogenic potential could be detected in this test

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative