4-oxo-4-(p-tolyl)butyric acid adduct with 4-ethylmorpholine

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:

22.02.1995 - 14.03.1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

419-240-6

EC Name:

-

Cas Number:

171054-89-0

Molecular formula:

Hill formula: C28H37NO7 CAS formula: (C11H12O3)2.C6H13NO

IUPAC Name:

bis(4-(4-methylphenyl)-4-oxobutanoic acid); 4-ethylmorpholine

Test material form:

solid: crystalline

Details on test material:

Sponsor's identification: TKA 40074 (CGCI 1405)
cream coloured
2:1 adduct of Benzenebutanoic acid, 4-methyl-gamma-oxo and 4-ethylmorpholine
batch No.: 1/94

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 50, 150, 500, 1500 and 5000 µg/plate
Concentration range in the main test (without metabolic activation): 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

Solvent: Dimethylsulfoxide

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene (with S9 only)

Details on test system and experimental conditions:

Tester Strains:
Salmonella typhimurium TA1535, TA1537, TA98 and TA100
Escherichia coli WP2uvrA
These strains were obtained from the British Industrial Biological Research Association on 14 August 1987 and were stored at -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34. Prior to being used, characterisation checks were carried out to determine the amino-acid requirement, presence of rfa, R factors, uvr mutation and the spontaneous reversion rate.
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37 °C for approximately 10 hours.

Preparation of Test and Control Materials:
The test material was accurately weighed and approximate half-log dilutions in dimethyl sulphoxide prepared on the day of each experiment. Analysis for concentration, homogeneity and stability of the test material formulations is not a requirement of the test guidelines and was, therefore, not determined.
Vehicle and positive controls were used in parallel with the test material. A solvent treatment group was used as the vehicle control.
In addition, the material 2-Aminoanthracene (2AA), which is non-mutagenic in the absence of metabolising enzymes was used in the S9 series of plates at different concentrations.

Microsomal Enzyme Fraction:
S9 was prepared in-house on 17/01/95. It was prepared from the livers of male Sprague-Dawley rats weighing ~ 200 g. These had each received a single i. p. injection of Aroclor 1254 at 500 mg/kg, 5 days before S9 preparation.
The S9 was stored at -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.

S9-Mix and Agar:
The S9-Mix was prepared at 4 °C as follows:
S9 5.0 mL
1.65 M KCl/0.4 M MgCl2 1.0 mL
0.1 M Glucose-6-phosphate 2.5 mL
0.1 M NADP 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL
Sterile distilled water 14.5 mL
Top agar was prepared using 0.6% Difco Bacto agar and 0.5% sodium chloride with 5 mL of 1.0 mK histidine/1.0 mO biotin and 1.0 mM tryptophan solution added to each 100 mL of top agar. Base agar plates were prepared using 1.2% Oxoid Agar Technical No. 3 with Vogel-Bonner Medium E and 20 mg/mL D-glucose.

Preliminary Toxicity Study:
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material to the tester organisms. A mixture of 0.1 mL of bacterial suspension (TA100 or WP2uvrA-), 0.1 mL of test solution, 0.5 mL phosphate buffer and 2 mL of molten, trace histidine/tryptophan supplemented media was overlaid onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). Five doses of the test material and a vehicle control (dimethyl sulphoxide) were tested in duplicate. After approximately 48 hours incubation at 37 °C the plates were scored for revertant colonies and examined for a thinning of the background lawn.

Mutation Study - Experiment 1 (Range-finding Study):
Five concentrations of the test material were assayed in triplicate against each tester strain, using the direct plate incorporation method in accordance with the standard methods for mutagenicity tests using bacteria.
Test Material and Vehicle Controls:
Known aliquots (0.1 mL) of one of the bacterial suspensions were dispensed into sets of sterile test tubes followed by 2.0 mL of molten trace histidine/tryptophan supplemented top agar at 45 °C, 0.1 mL of the appropriately diluted test material or vehicle control and either 0.5 mL of the S9 liver microsome mix or 0.5 mL of pH 7.4 buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material with and without S9-mix.
Positive Controls:
Without Activation: A known aliquot (0.1 mL) of one of the positive control solutions (ENNG, 9AA or 4NQ0) was added to a test tube containing 2.0 mL of molten, trace histidine/tryptophan supplemented top agar and 0.1 mL of the appropriate bacterial suspension. Finally, 0.5 mL of pH 7.4 buffer was added to the tube, the contents mixed and poured onto an agar plate. This procedure was then repeated, in triplicate, for each tester strain.
With Activation: A known aliquot (0.1 mL) of 2 AA solution was added to a test tube containing 2.0 mL of molten, trace histidine/trpytophan supplemented top agar and 0.1 mL of the appropriate bacterial suspension. Finally, 0.5 mL of S9-mix was added to the tube, the contents mixed and poured onto an agar plate. This procedure was then repeated, in triplicate, for each tester strain.
The plates were incubated at 37 °C for approximately 48 hours and the number of revertant colonies counted.

Mutation Study - Experiment 2 (Main Study):
The secon experiment was performed using methodology as described for experiment 1, using fresh bacterial cultures, test material and control solutions in triplicate.

Interpretation of Results:
For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. If the two experiments give conflicting results or equivocal results are obtained, then a third experiment may be used to confirm the correct response. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 μg/plate. In this case the limiting factor was the maximum recommended dose.

Results and discussion

Test results

Test results 1

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

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

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 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 4

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 5

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

Additional information on results:

Preliminary Toxicity Study:
The dose range of the test material used in the preliminary toxicity study was 0, 50, 150, 500, 1500 and 5000 μplate. The test material was non-toxic in the strains of bacteria used (TA100 and WP2uvrA-).
Mutation Study:
The results of the checks for characteristics, viability and spontaneous reversion rate for each tester strain were all found to be satisfactory.
No toxicity was exhibited to any of the strains of bacteria used.
No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material either with or without metabolic activation.
All of the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies and the activity of the S9 fraction was found to be satisfactory.

Applicant's summary and conclusion

Conclusions:

The test material was found to be non-mutagenic under the conditions of this test.

Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA^- were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). This method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MOL and MAFF. It also meets the requirements of the OECD, EC, and USE, EPA (TSCA) guidelines. The dose range was determined in a preliminary toxicity assay and was 50 to 5000 μg/plate in the first experiment. The experiment was repeated on a seperate day using the same dose range as experiment 1, fresh cultures of the bacterial strains and fresh chemical formulations.

The vehicle (dimethyl sulphoxide) control plates produced counts of revertant colonies within the normal range.

All of the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies, both with and without the metabolising system.

The test material caused no visible reduction in the growth of the bacterial lawn at any dose level either with or without metabolic activation, it was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate.

No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material, either with or without metabolic activation. The test material was found to be non-mutagenic under the conditions of this test.