Dimethylammonium 2,4-dichlorophenoxyacetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions Impurities of the test substance are not reported

Data source

Reference

Reference Type:

publication

Title:

Evaluation of the genotoxicity of 2,4-dichlorophenoxyacetic acid and its derivatives in mammalian cell cultures

Author:

Gollapudi, B.B., Charles, J.M., Linscombe, V.A., Day, S.J., Bus, J.S.

Year:

1999

Bibliographic source:

Mutation Research, 444, 217-225

Materials and methods

Test guideline

Qualifier:

no guideline followed

Deviations:

not applicable

Principles of method if other than guideline:

The assay was conducted by methods described by O'Neill and Hsie (1979) and O'Neill et al. (1977, 1982)

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

5742-17-6

Cas Number:

5742-17-6

IUPAC Name:

5742-17-6

Constituent 2

Reference substance name:

isopropylamine 2,4-dichlorophenoxyacetate

IUPAC Name:

isopropylamine 2,4-dichlorophenoxyacetate

Constituent 3

Reference substance name:

2,4-D, isopropylamine salt

IUPAC Name:

2,4-D, isopropylamine salt

Details on test material:

- Name of test material (as cited in study report): 2,4-D IPA
- Analytical purity: 50.15% of active ingredient

Method

Target gene:

HGPRT locus

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

used cells: CHO-K1-BH4

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (Sprague-Dawley rat liver, Aroclor 1254- induced); final concentration in the treatment medium = 2% v/v

Test concentrations with justification for top dose:

0, 500, 1000, 1500, 2000 and 3000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO or distilled water
Final concentration of the solvent in the treatment medium: 1%

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 20-methylcholanthrene (4 µg/mL)

Remarks:

without S9-mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

with S9-mix

Migrated to IUCLID6: 621 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4-5 h
- Selection time (if incubation with a selection agent): 7-9 days

SELECTION AGENT (mutation assays): 6-thioguanine (10 µg/mL)

NUMBER OF CELLS EVALUATED: The frequency of mutants per 10 to the power of 6 clonable cells

DETERMINATION OF CYTOTOXICITY
- Cloning efficiency was estimated by plating 200 cells/60 mm plate (three plates/replicate).
The test material toxicity was assessed by determining the cloning efficiency of the cells at the end of the treatments and expressed relative to the negative controls (relative cell survival, RCS)

Evaluation criteria:

The frequency of mutants per 10 to the power of 6 clonable cells was statistically evaluated using a weighted analysis of variance.

Statistics:

A linear trend test and lack of fit test was employed (α = 0.05) as an omnibus test to compare treated groups to the negative control. If there was a significant increasing trend or a significant lack of fit, a Dunnett's test was conducted, comparing each group and the positive control to the negative control (α = 0.05, one-sided). An additional comparison of the positive control to the negative control was conducted using a linear contrast statement.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

for the highest dose (3000 µg/mL) without activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Results of CHO/HGPRT assay on 2,4-D IPA

Concentration (µg/mL)	S9 (+/-)	Assay 1		Assay 2
		RCS (%)	MF (x 10^-6)	RCS (%)	MF (x 10^-6)
0	-	98.6	1.5	95.4	0.9
	-	101.4	1.8	104.6	0.0
	+	110.8	2.4	99.3	0.0
	+	89.2	0.8	100.7	5.7
500	-	100.1	2.7	128.3	1.4
	-	94.4	0.0	137.8	5.8
	+	92.4	3.1	88.0	11.3
	+	89.2	2.1	96.6	5.0
1000	-	86.3	4.5	139.4	9.0
	-	102.9	4.4	134.5	9.1
	+	90.0	4.5	81.2	5.3
	+	97.2	6.9	78.2	5.3
1500	-	86.3	3.7	125.5	5.5
	-	87.3	2.5	139.4	11.6
	+	88.4	3.5	82.8	6.1
	+	101.5	6.5	78.2	1.5
2000	-	73.9	4.0
	-	73.1	2.2	97.5	10.1
	+	106.0	4.9	82.8	6.7
	+	90.6	9.7
3000	-	46.9	6.2	36.6	*
	-	43.2	0.0	40.6	14.7
	+	64.8	7.3	61.7	13.2
	+	68.8	3.6	20.0	3.6
PC	-	45.2	157.5	56.6	298.2
	-	35.4	264.5	55.1	350.9
	+	75.7	90.3.	100.7	113.0
	+	66.9	106.9	86.8	127.9

RCS: relative cell survival following treatment;

MF: mutant frequency; PC: positive control

* culture lost due to impaired cell proliferation

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative