Propanoic acid, 2-(4-chloro-2-methylphenoxy)-, 2-ethylhexyl ester, (2R)-

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:

July 7, 1992 - July 31, 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only 4 S. typhimurium strains were used

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

other: EEC Directive 79/831/EEC, Annex V (Directive 84/449/EEC), Method B.14: Salmonella typhimurium- Reverse Mutation Assay

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

other: US EPA, Method: HG-Gene Muta (40CFR798.5265)-S.typhimurium; The Salmonella typhimurium reverse mutation assay

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-Ethylhexyl (R)-2-(2-methyl-4chlorophenoxy)propionate

EC Number:

630-324-3

Cas Number:

861229-15-4

Molecular formula:

C18H27ClO3

IUPAC Name:

2-Ethylhexyl (R)-2-(2-methyl-4chlorophenoxy)propionate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material: 5007
- sample No.:1505E
- Expiration date of the lot/batch: March 1993

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 4°C in the dark

Method

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Professor B.N. Ames, University of California, U.S.A.
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Nutrient broth (DAB 7, Merck)
- Properly maintained: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

other: deficient in normal DNA repair processes; strains TA 98 and 100 possess a plasmid (pKM101) which introduces an error-prone repair process, resulting in increased sensitivity to mutagens

Cytokinesis block (if used):

no

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from rat liver stimulated with Aroclor 1254

Test concentrations with justification for top dose:

50, 150, 500, 1500 and 5000 µg/plate
At the highest dose of 5000 µg/plate no toxicity was observed in the preliminary dose range finding study. This dose is recommended by the regulatory guidelines with which this study complies.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Prior to commercing testing the solubility of the test material in ethanol was assessed. The test material was found to be soluble in ethanol at 50 mg/mL so ethanol was used as the solvent for the assay.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding: 2x10E9 cells per mL

DURATION
- Preincubation period:
An aliquot of 0.1 mL of a 10 h bacterial culture and 0.5 mL S9-Mix or 0.1 M phoshate buffer (pH 7.4) were placed in glass bottles. An aliquot of 0.1 mL of the test solution was added, followed immediatly by 2 mL of histidine deficient agar. The mixture was overlaid onto previously prepared petri dishes containing 25 mL minimal agar. Three petri dishes were used for each dose level. A set of plates were also prepared containing only bacterial culture and S9-Mix or phosphate buffer (0 µg/plate). Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9-Mix and phosphate buffer. The strains were also tested for the presence of the following genotypic markers: rfa,uvrB and pKM101. Plates were incubated at 37°C for 3 days. After this period revertant colonies were counted.

Evaluation criteria:

For the test to consider valid, the following criteria must have been met:
- the presence of the relevant genotypic markers should have been confirmed
- the mean revertant colony counts for the solvent control for each strain should have been within the ranges HRC´s historical data base:
TA 1535 5-20
TA 1537 5-20
TA 98 15-40
TA 100 70-160

The mean number of reverant colonies for all treatment groups was compared with those obtained for the solvent controls.

Statistics:

A test substance is considered to show evidence of mutagenic activity if a reproducible statistically significant increase (p<0.05) in revertant colony numbers is observed. The statistical procedures are those described by Mahon et al. (1989) and analysis of variance followed by Dunnett´s test was used.

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

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

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

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
No visible effect on the background bacterial lawn or decrease in revertant colony numbers was observed at any dose level in either the presence or absence of S9-Mix in a preliminary toxicity test. No precipitation of the test material up to the highest concentration used was observed.

Any other information on results incl. tables

The genotypic markers rfa, uvrB and pKM101 were shown to be present in the appropriate strains in both mutation tests. All the revertant colony counts for the solvent controls were within the stated ranges for a valid assay. The test substance was stable in the solvent for at least 4 h. The test substance stock solution was analyzed for each test for achieved concentration and was found to be within 3% of the stated concentration of the active ingredient in all tests.

Applicant's summary and conclusion

Conclusions:

The test substance MCPP-P 2-EHE shows no evidence of mutagenic activity in the bacterial system.

Executive summary:

A study similar to OECD 471 was performed to assess the in vitro mutagenic potential of MCPP-P 2-EHE using histidine-dependent auxotrophic mutants of S.typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100). Strains were exposed to the test material diluted in ethanol. In a preliminary dose range finding study no toxicity was observed up to the highest concentration of 5000 µg/plate. Five concentrations (50, 150, 500, 1500 and 5000 µg/plate) were used in the plate incorporation mutation assay. Bacteria were incubated with the test substance or solvent control ethanol in the presence or absence of rat liver S9-Mix for 3 days. No evidence of mutagenic activity was seen at any dose level of MCPP-P 2-EHE. The concurrent positive control compounds demonstrated the sensitivity of the assay and metabolizing activity of the liver S9-Mix.