Methyl O-(4-amino-3,5-dichloro-6-fluoropyridin-2-yloxy)acetate

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions No guideline statement, but in general accordance with OECD guideline 476, which was set in force later. Preparation of S-9 mix and evaluation criteria were not reported.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1983

Report date:

1983

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

; Preparation of S-9 mix and evaluation criteria were not reported.

GLP compliance:

no

Remarks:

GLP was not compulsory during conduct of study. However, the study was performed according to GLP standards.

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Methylheptyl, 0-(4-amino-3,5-dichloro-6-fluoro-2-pyridyloxy) acetate

IUPAC Name:

Methylheptyl, 0-(4-amino-3,5-dichloro-6-fluoro-2-pyridyloxy) acetate

Constituent 2

Reference substance name:

Acetic acid [(4-amino-3,5-dichloro-6-fluoro-2-pyridinyl) oxy], 1-methylheptyl

IUPAC Name:

Acetic acid [(4-amino-3,5-dichloro-6-fluoro-2-pyridinyl) oxy], 1-methylheptyl

Constituent 3

Reference substance name:

1-methylheptyl [(4-amino-3,5-dichloro-6-fluoropyridin-2-yl)oxy]acetate

EC Number:

279-752-9

EC Name:

1-methylheptyl [(4-amino-3,5-dichloro-6-fluoropyridin-2-yl)oxy]acetate

Cas Number:

81406-37-3

IUPAC Name:

1-methylheptyl [(4-amino-3,5-dichloro-6-fluoropyridin-2-yl)oxy]acetate

Details on test material:

- Name of test material (as cited in study report): Dowco 433
- Physical state: Pinkish powder
- Purity: not reported
- Lot/batch No.: 433T-1282-7
- Stability: not reported

Method

Target gene:

hypoxanthine-guanine phosphoribosyl transferase gene

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The cells were maintained in Ham´s F10 medium supplemented with newborn calf serum (15%) and glutamine (2 mM). Streptomycin (50 mg/mL) and penicillin G (50 mg/L) are additionally added to the medium used in the toxicity and point mutation tests. Incubation conditions were 37 °C in an atmosphere of 5% CO2 / 95% air (v/v). Cells were passaged twice weekly after trypsinization with a split ratio of 1:10.

Additional strain / cell type characteristics:

other: sub-line CHO-K1, ATCC no. CCL 61

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix from livers of Aroclor-1254-treated male rats (500 mg/kg bw), obtained from CIVO Toxicology and Nutrition TNO, Zeist, The Netherlands.

Test concentrations with justification for top dose:

Preliminary cytotoxicity assay: 0, 100, 250, 500, 750, 1000, 1500 and 2000 µg/mL

Mutation assays: 0, 500, 1000, 1500, 1750 and 2000 µg/ml

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

Migrated to IUCLID6: 2 µL/mL with activation

Controls 2

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Migrated to IUCLID6: 0.4 µL/mL without activation

Details on test system and experimental conditions:

see any other information on materials and methods incl. tables

Evaluation criteria:

see any other information on materials and methods incl. tables

Statistics:

Not reported.

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:

signs of cytotoxicity were observed at dose levels of > 1500 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: Chinese hamster Ovary (CHO)

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

CYTOTOXICITY ASSAY

Cytotoxicity was evident at concentrations of 1500 µg/mL and above, both with and without metabolic activation (see Table 1) Based on the results of this test the following concentrations were used for the mutation test: 0, 500, 1000, 1500, 1750 and 2000 µg /ml.

 

Table 1: Result of cytotoxicity test with Dowco 433 inCHO cells

Concentration	With S-9 mix (5 h exposure)		Without S-9 mix (24 h exposure)
	Clonable cells	rel. survival	Clonable cells	rel. survival
[µg/mL]	[mean of 3 plates]	[%]	[mean of 3 plates]	[%]
0	802	100	810	100
100	844	105	821	101
250	819	102	793	98
500	776	97	785	97
750	724	90	760	94
1000	698	87	726	90
1500	595	74	560	69
2000	298	37	518	64

 

 

POINT MUTATION TEST

 

No concentration-dependent increases in the mutation frequency were observed, both with and without metabolic activation (see Table 2).

The increase of the mutation rate observed at 1000 µg/mL in the second experiment without metabolic activation was not considered test substance related, since it was not confirmed at higher doses or in the other experiments.

The positive control substances, EMS and DMN, gave the expected increases in the mutation frequency.

 

Table 2: Results of point mutation test with Dowco 433 at the HGPRT-locus of CHO cells

	1^stexperiment			2^ndexperiment
Test substance concentration [µg/mL]	Mean absolute initial survival [%]	Mean absolute final survival [%]	Mutation frequency [no/10^6clonable cells]	Mean absolute initial survival [%]	Mean absolute final survival [%]	Mutation frequency [no/10^6clonable cells]
Without metabolic activation (24 h exposure)
0	86	88	9	71	91	6
500	64	83	9	63	77	11
1000	54	92	14	56	64	16
1500	52	89	10	55	90	10
1750	53	82	13	49	93	9
2000	45	91	13	42	81	7
EMS (0.4 µL/mL, 5h exposure)	36	80	872	39	82	640
With metabolic activation (5 h exposure)
0	63	86	9	75	82	14
500	69	90	11	73	83	10
1000	64	83	7	65	78	8
1500	52	99	15	62	73	13
1750	48	86	15	50	79	8
2000	20	84	9	41	65	13
DMN (2.0 µL/mL)	17	76	240	14	56	364

     EMS= Ethyl methylsulfonate

     DMN = Dimethylnitrosamin

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Based on the study results Dowco 433 does not induce point mutations at the HGPRT locus of CHO cells in vitro.

Executive summary:

The potential of Dowco 433 to induce mutations at the HGPRT locus of Chinese Hamster ovary cells, was tested, both with and without a metabolic activation system (S-9 mix from the livers of Aroclor 1254-induced male rats).

No test-substance related increases in the mutation frequencies were observed, either with or without metabolic activation. The positive control substances, Dimethylnitrosamin (with S-9) and Ethyl methylsulfonate (without S-9 mix), showed both a clear increase in the mutation frequency. Thus, Dowco 433 does not induce point mutations at the HGPRT locus of CHO cells in vitro.