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Toxicological information

Genetic toxicity: in vitro

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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 guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only 4 S. typhimurium strains were used
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
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

Constituent 1
Chemical structure
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 resultsopen allclose all
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
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
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
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.