Methyl (R)-2-(4-(3-chloro-5-trifluoromethyl-2-pyridyloxy)phenoxy)propionate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EEC: Directive 97/69/EEC, of July 31, 1992 Adapting to Technical Progress for the Seventeenth Time Council Directive 67/548/EEC, EEC Publication No. L383A, 1992

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Methyl (R)-2-(4-(3-chloro-5-trifluoromethyl-2-pyridyloxy)phenoxy)propionate

EC Number:

406-250-0

EC Name:

Methyl (R)-2-(4-(3-chloro-5-trifluoromethyl-2-pyridyloxy)phenoxy)propionate

Cas Number:

72619-32-0

Molecular formula:

C16H13ClF3NO4

IUPAC Name:

methyl 2-(4-{[3-chloro-5-(trifluoromethyl)pyridin-2-yl]oxy}phenoxy)propanoate

Test material form:

solid

Specific details on test material used for the study:

Haloxyfop-R Methyl Ester Technical
Lot #: NB10150101 (TSN101748)
Purity: 98.4%

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system
- Source of S9 : Molecular Toxicology, Inc.
- Method of preparation of S9 mix: The homogenate was prepared from male Sprague-Dawley rats that had been injected (i.p.) with Aroclor™ 1254 (200 mg per ml in com oil) at 500 mg/kg.
- Concentration or volume of S9 mix: 1.00 mL

Test concentrations with justification for top dose:

Dose range finding study: 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3330, 5000 µg/plate
Mutagenicity study: 33.3, 100, 333, 1000, 3330, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: At 100 mg per mL, which was the most concentrated stock dilution prepared, the test article formed a transparent, beige solution. The test article remained a solution in all succeeding dilutions prepared for the mutagenicity assay.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene: TA100, TA1535, TA1537 (2.5 µg; with S9); WP2uvrA (25.0 µg; with S9), ICR-191: TA1537 (2.0 µg; without S9)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: Two (initial mutagenicity assay and confirmatory assay)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium: Preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 ± 2 minutes
- Exposure duration/duration of treatment: 52 ± 4 hour


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Background growth inhibition

Evaluation criteria:

For a test substance to be considered positive, it had to produce at least a 3-fold (TA98, TA1535, TA1537, and WP2uvrA) or 2-fold (TA100) dose related and reproducible increase in the mean revertants per plate of at least one tester strain over the mean revertants per plate of the appropriate vehicle control. An observed response which did not meet all three of the above criteria (magnitude, dose-responsiveness, reproducibility) was not evaluated as positive.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Indications of cytotoxicity were observed with tester strain TA100 with S9 mix at 3330 µg/plate and above as evidenced by a slight reduction of the bacterial background lawn.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: A dose range finding study was conducted on the test article using tester strains TA100 and WP2uvrA in both the presence and absence of S9 mix (one plate per dose). Ten doses of test substance, from 5000 to 6.67 µg per plate, were tested. Indications of cytotoxicity were observed with tester strain TA100 in the presence of S9 mix at 3330 ug per plate and above as evidenced by a slight reduction of the bacterial background lawn. No cytotoxicity was observed with tester strain TA100 in the absence of S9 mix or with WP2uvrA in either the presence or absence of S9 mix as evidenced by a normal background lawn and no decrease in the number of revertants per plate. Slight article precipitate was observed at 3330 µg per plate and above in the presence of S9 mix and at 1000 and 3330 µg per plate in the absence of S9 mix. Moderate test article precipitate was observed at 5000 µg per plate in the absence of S9 mix.

Any other information on results incl. tables

Table-1: Mutagenicity assay results (mean revertants per plate)

Conc. (µg/plate)	TA1537		TA1535		TA98		TA100		WP2uvrA
	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9
DMSO	6	12	7	9	9	24	96	93	17	13
33.3	3	8	8	10	15	30	79	94	16	14
100	4	9	9	13	9	20	100	98	20	12
333	8	9	12	12	13	26	106	101	14	14
1000	8	9	11	12	9	20	109	80	14	15
3330	9	11	8	9	9	16	96	70	13	12
5000	9	8	7	12	11	20	98	71	9	15
Positive cotnrol	1678	168	588	107	178	500	623	787	451	205

Table-2: Confirmatory assay results (mean revertants per plate)

Conc. (µg/plate)	TA1537		TA1535		TA98		TA100		WP2uvrA
	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9
DMSO	7	7	10	11	14	26	92	96	17	15
33.3	8	12	10	10	11	28	87	101	14	16
100	4	10	17	13	16	21	90	96	14	16
333	7	10	11	10	13	25	91	86	17	12
1000	9	5	10	8	11	22	84	103	15	14
3330	7	8	10	11	12	19	89	85	14	15
5000	7	6	15	10	9	26	102	74	18	13
Positive cotnrol	1898	153	710	108	243	423	673	979	547	258

Applicant's summary and conclusion

Conclusions:

Negative in reverse bacterial mutation test

Executive summary:

The test substance was evaluated in the Salmonella-Escherichia coli/mammalian-microsome reverse mutation assay following OECD guieline 471 and U S EPA OPPTS 870.5100. The tester strains used in this study were Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537, and Escherichia coli tester strain WP2uvrA. The assay was conducted using 6 doses of test substance in both the presence and absence of an externally supplied metabolic activation system (S9) along with concurrent vehicle and positive controls. The concentration of the test material ranged from 33.3 to 5000 µg/plate in the presence and absence of metabolic activation. The results of the initial mutagenicity assay were confirmed in an independent experiment. The test material did not induce a positive increase in the number of revertants per plate of any of the tester strains either in the presence or absence of microsomal enzymes (S9). Hence the test substance was classified as negative in this bacterial mutagenicity test under the experimental conditions used.