Mecoprop

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:

18 February 1981 to 09 March 1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1981

Report date:

1981

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Remarks:

Study pre-dates GLP

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Mecoprop

EC Number:

230-386-8

EC Name:

Mecoprop

Cas Number:

7085-19-0

Molecular formula:

C10H11ClO3

IUPAC Name:

2-(4-chloro-2-methylphenoxy)propanoic acid

Details on test material:

- Storage: Stored at 4 °C

Method

Target gene:

Histidine requirement in Salmonella typhimurium strains.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain 2

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: Male Sprague-Dawley rats, 200-300 g
- Method of preparation of S9 mix: To activate the enzymes which metabolise foreign substances, the rats received a single intraperitoneal injection of 500 mg Aroclor 1254 per kg body weight 5 days before sacrifice. On the fifth day the rats were killed, and the livers prepared. The livers were weighed and washed in an equivalent volume of a 150 mM KCl solution (1 mL KCl to 1 g wet Liver), then cut into small pieces and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 g for 10 minutes at +4 °C, 5 mL portions of the supernatant (so-called S-9 fraction) are quickly deep-frozen in dry ice and stored at -70 °C to -80 °C for 2 months at the most.
- Concentration or volume of S9 mix and S9 in the final culture medium: 3 volumes of S-9 fraction are mixed with 7 volumes of S-9 supplement (cofactors). Concentration of co-factors: MgCL 8 mM, KCl 33 mM, glucose-6-phosphate 5 mM, NADP 4 mM, phosphate buffer (pH 7.4) 100 mM.

Test concentrations with justification for top dose:

100, 500, 2500 and 5000 µg/plate.

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

N-ethyl-N-nitro-N-nitrosoguanidine

other:

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Four test plates for each concentration

METHOD OF TREATMENT/ EXPOSURE:
- Test tubes containing 2 mL portions of soft agar which consists of 100 mL agar (0.6 % agar + 0.6 % NaCl) and 10 mL amino-acid solution (minimal amino-acid solution for the determination of mutants: 0.5 mM histidine and 0.5 mM biotin) are kept in water at 45 °C, and the remaining components are added in the following order: 0.1 mL test solution , 0.1 mL bacterial suspension, 0.5 mL S-9 mix (in tests with metabolic activation) or 0.5 mL phosphate buffer (in tests without metabolic activation) After mixing, the samples are poured onto the nutrient plates within approx. 30 seconds.

FOR GENE MUTATION:
- Expression time: After incubation for 48 hours at 37 °C in the dark, the bacterial colonies (his+ revertants) are counted.
- Method used: Agar overlay

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition and reduction in the number of revertant colonies

Evaluation criteria:

In general, a substance to be characterised as positive in the Ames test has to fulfill the following requirements: doubling of the spontaneous mutation rate (control), dose-response relationship and reproducibility of the results.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At highest dose 5 000 µg/plate, led to reduced his- background growth

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Withought S-9 mix, slight decrease in the number of revertant colonies at highest dose level of 5 000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS:
- Tests without S-9 mix: Without metabolic activation the test material showed no mutagenicity. The number of his+ revertants was always in the range of that of the control for all strains. The highest dose of 5 000 µg/plate showed a slightly toxic effect and, in some cases, led to a reduced his- background growth (TA 1537) or to a slight decrease in the number of revertant colonies (TA 98)
- Tests with S-9 mix: After metabolic activation no mutagenic effect was detected, the number of revertant colonies was again in the same range as that of the control. Under these experimental conditions, too, a slightly toxic effect was detected at the highest dose level (reduced his- background growth or slight decrease in the number of his+ revertants).

Any other information on results incl. tables

Study Results

+/- S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		TA1535	TA1537	TA1538	TA98	TA100
+	PC Vehicle 20 100 500 2500 5000	376 20 16 15 14 11 12	105 12 13 11 10 4 B	1408 27 26 22 25 20 11	1623 40 40 38 36 31 23	2175 103 117 112 117 113 111
-	PC Vehicle 20 100 500 2500 5000	2263 16 16 14 13 13 14	503 7 8 9 6 5 B	264 14 15 15 11 12 10	790 20 19 19 19 17 14	2250 117 122 124 136 122 117

Mean number of revertant colonies/4 replicate plates

PC = Positive control

B= reduced his- background growth

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test material was not mutagenic.

Executive summary:

The genotoxicity of the test material was investigated, according to a similar method described in OECD Test Guideline 471.

The test material was tested for mutagenicity in the Ames test in a dose range of 20 µg - 5 000 µg/plate using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100. The test was carried out directly and in the presence of a mammalian metabolising system (9 000 g supernatant of liver homegenate from rats treated with Aroclor 1254, the so-called S-9 mix) in order to register possible mutagenic metabolites. Under all these experimental conditions no mutagenicity was detected for the test material. The number of his+ revertants was always in the range of that of the control, both using the base pair strains TA 1535 and TA 100 and using the frameshift strains TA 1537, TA 1538 and TA 98. The highest dose of 5000 µg/plate was slightly toxic for some strains and, in some cases, led to a reduced his-­ background growth or to a slight decrease in the number of mutant colonies.

Under the conditions of this study, the test material was not mutagenic.