mecoprop-P (ISO); (R)-2-(4-chloro-2-methylphenoxy)propionic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 February 1990 to 23 February 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA FIFRA Guidelines

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(R)-2-(4-chloro-2-methylphenoxy)propionic acid

EC Number:

240-539-0

EC Name:

(R)-2-(4-chloro-2-methylphenoxy)propionic acid

Cas Number:

16484-77-8

Molecular formula:

C10H11ClO3

IUPAC Name:

(R)-2-(4-chloro-2-methylphenoxy)propionic acid

Test material form:

solid: flakes

Remarks:

Brown flakes

Details on test material:

- Storage conditions: Stored at room temperature and protected from light until required.

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9 : Young male CD rats, ca. 200 g bodyweight, were obtained from Charles River Breeding Laboratories (U.K.), Margate, Kent. Aroclor 1254 (500 mg/kg bodyweight in corn oil) was administered as a single intraperitoneal injection to induce microsomal enzyme activity. Four days after treatment, the animals were fasted overnight and then killed by cervical dislocation.
- Method of preparation of S9 mix: The livers were removed, washed in cold 0.15M KCl, then homogenised with more of the same medium (approximately 3 mL per g wet liver) in a Potter-Elvehjem homogeniser. Homogenates were centrifuged at 9 000 g for 10 minutes and supernatants collected and stored at -80 °C until required for preparation of the S-9 mix. Supernatant was used within 3 months of preparation.

S9-mix was prepared using:
0.4 mL 0.1 M NADP, sodium salt in aqueous solution
0.5 mL 0.1 M glucose-6-phosphate, sodium salt in aqueous solution
0.2 mL 0.4 M MgCl2·6H2O/1.65 M KCl aqueous solution
1.5 mL supernatant from liver homogenate
10 mL 0.1 M KH2PO4-Na2HPO4 buffer (pH7.4)

Test concentrations with justification for top dose:

Main Experiment: 10, 32, 100, 316 and 1 000 µg/plate (following preliminary toxicity test).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

benzo(a)pyrene

other: 2-Aminoanthracene 5 µg/plate in DMSO, with and without S9 mix, strain TA1535; 2-nitrofluorene 5 µg/plate in DMSO, without S9 mix, strain TA98.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments : Each test, in each strain, was conducted on two separate occasions.

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added: Plate incorporation
- Pour-plate assay
An aliquot (0.1 mL) of each concentration of the test material was placed in a sterile tube. Molten, histidine-deficient top-agar (2 mL) and bacterial suspension (0.1 mL), maintained at 45 °C, was then added. The tubes were mixed by inversion and 0.5 mL rat liver microsomal preparation (S-9 mix) was added where appropriate. The tubes were again inverted to mix thoroughly and the contents poured onto plates containing solidified minimal medium (20 mL). Further plates were prepared without the inclusion of the test organisms to verify the sterility of the S-9 mix and the test material. A control series of plates was prepared to confirm the inability of DMSO (0.1 mL) to induce reversion in the bacterial strains, and to provide a measure of the spontaneous mutation rates.
Aliquots (0.1 mL) of a 10^-6 dilution of culture were spread on the surface of plates of complete medium to measure the viability and cell-density of each culture.
All plates were prepared in triplicate, allowed to solidify and incubated at 37 °C for 2 days. After incubation, numbers of revertant colonies were counted, either manually or with a Biotran II automatic colony counter. Total colonies on nutrient plates were counted in the same way. Growth of the background lawn of non-revertant cells on minimal plates was verified.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Background growth inhibition
- Preliminary Toxicity Test: A solution of the test material was prepared in DMSO at 25 mg/mL, and an aliquot of this solution (0.2 mL) was transferred to a sterile tube containing molten, histidine-deficient top-agar (2.0 mL) maintained at 45 °C. An additional aliquot (0.1 mL) of the test material solution was similarly transferred to another tube of molten top-agar (2.0 mL).
Three serial ten-fold dilutions in molten top-agar were prepared from each of these preparations, giving a series of eight different concentrations of test material from 2.5 µg to 5 mg per plate. All tubes were inoculated with an overnight culture of strain TA98 (0.1 mL) and overlaid onto minimal medium plates. Control plates were prepared containing top-agar and culture alone, top-agar, DMS0 (0.2 mL) and culture, and top-agar and test material (0.1 mL) without bacterial culture.
The plates were incubated at 37 °C for 2 days and were then examined for the presence of a background lawn of non-revertant colonies; toxicity of the test material is shown by absence or thinning of the background lawn. The level of test material chosen as the top level for pour-plate tests is normally the lowest level causing visible thinning of the lawn. (In the absence of such thinning, a top level of 5 mg/plate would be selected):
The control plates were checked for the absence of growth on sterility checks or normal counts on negative controls in the presence and absence of the solvent.

Rationale for test conditions:

Based on results of preliminary toxicity test.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- Preliminary toxicity test: The background lawn of non-revertant cells was absent following exposure to the test material at 2 500 µg per plate. A normal background lawn and revertant colony count was obtained with the test material at 500 µg per plate. A top exposure level of 1 000 µg per plate was therefore selected for use in the main assays.
- Sterility checks, spontaneous reversion rate and viability checks: The absence of colonies on the test material and S-9 mix sterility check plates indicates that these preparations were free of significant microbial contamination.
The total colony counts on plates number 18 confirmed the viability and high cell density of the cultures of the individual organisms. The counts recorded on appropriate negative control plates confirmed the characteristically low spontaneous reversion rates of the tester strains and the absence of effects on these rates of DMSO inclusion.
- Mutagenic activity of positive control chemicals: Appropriate positive control chemicals (with S-9 mix where required) induced marked increases in revertant colony numbers with all strains, confirming sensitivity of the cultures and activity of the S-9 mix.
- Action of the test material: No increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test material at levels from 10 to 1 000 µg per plate. Inhibition of growth, observed as thinning of the background lawn of non-revertant cells, occurred in all strains following exposure to the test material at 1 000 µg per plate.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the test material was devoid of mutagenic activity.

Executive summary:

The test material was examined for mutagenic activity in four histidine-dependent auxotrophs of Salmonella typhimurium, strains TA98, TA100, TA1535 and TA1537, using pour-plate assays according to OECD Test Guideline 471 and in compliance with GLP. Each test, in each strain, was conducted on two separate occasions.

The studies, which were conducted in the absence and presence of an activating system derived from rat liver (S-9 mix), employed a range of levels of the test material from 10 to 1 000 µg per plate, selected following a preliminary toxicity test in strain TA98. All tests included solvent (dimethyl sulphoxide) controls with and without S-9 mix.

No increases in reversion to prototrophy were obtained with any of the four bacterial strains at the test material levels tested, either in the presence or absence of S-9 mix. Inhibition of growth, observed as thinning of the background lawn of non-revertant cells, occurred in all strains following exposure to the test material at 1 000 µg per plate.

Marked increases in the number of revertant colonies were induced by the known mutagens benzo[a]pyrene, 2-nitrofluorene, 2-aminoanthracene, 9-aminoacridine and sodium azide when examined under similar conditions.

Under the conditions of the study, the test material was devoid of mutagenic activity.