2-Propenoic acid, 2-methyl-, 2-(4-benzoyl-3-hydroxyphenoxy)ethyl ester

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in accordance with the OECD guideline 471 (adopted 21 July 1997) and in compliance with GLP (incl. certificate).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-benzoyl-3-hydroxyphenoxy)ethyl 2-methylprop-2-enoate

Cas Number:

16613-04-0

Molecular formula:

C19 H18 O5

IUPAC Name:

2-(4-benzoyl-3-hydroxyphenoxy)ethyl 2-methylprop-2-enoate

Details on test material:

- Name of test material (as cited in study report): ULS-611, Test substance No. 08/0140-1
- Physical state: solid, white
- Analytical purity: 99.2 g/100 g (analyzed by NMR method)
- Impurities (identity and concentrations): not specified
- Lot/batch No.: not indicated
- Storage condition of test material: refrigerator, light exclusion

Method

Target gene:

Salmonella typhimurium: Determination of the rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his+).
Escherichia coli: Determination of the rate of induced back mutations of bacterial mutant from trypthophan auxotrophy (trp-) to tryptophan prototrophy (trp+).

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from the liver of male Wistar rats (treated i.p. with 80 mg/kg bw phenobarbital and 80 mg/kg bw ß-naphthoflavone orally on 3 consecutive days), mixed with a series of cofactors (MgCl2, KCl, glucose-6-phosphate, NADP, phosphate buffer pH7.4).

Test concentrations with justification for top dose:

Standard plate test with and without S9-mix (all strains): 0, 20, 100, 500, 2500 and 5000 µg per plate
Preincubation test with S9-mix (Salmonella strains only): 0, 10, 50, 250, 1250 and 2500 µg per plate
Preincubation test with S9-mix (E. coli): 0, 20, 100, 500, 2500 and 5000 µg per plate
Preincubation test without S9-mix (all strains): 0, 20, 100, 500, 2500 and 5000 µg per plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO had been demonstrated to be a suitable vehicle in bacterial reverse mutation tests; historical control data are available for DMSO.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

. Historical negative control data for water, DMSO and acetone were given

Positive controls:

yes

Positive control substance:

other: see details on test system

Details on test system and experimental conditions:

METHOD OF APPLICATION:
A standard plate test and a preincubation test were conducted, both with and without metabolic activation (S9 mix). Each test was conducted in triplicates.

STANDARD PLATE TEST:
The experimental procedure of the standard plate test (plate incorporation method) is based on the method of Ames et al. (Mut. Res. 31: 347-364, 1975) and Maron and Ames (Mut. Res. 113: 173-215, 1983)
Salmonella typhimurium:
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42-45°C, and the remaining components are added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation).
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.
Composition of the minimal glucose agar:
980 mL purified water
20 mL Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose, monohydrate.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) are counted.

Escherichia coli:
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation).
After mixing, the samples are poured onto minimal agar plates within approx. 30 seconds.
The composition of the minimal agar (SA1 selective agar) is based on the description of Green, M.H.L. and Muriel, W.J. (Mut. Res. 38: 3-32, 1976), with the exception of solution E (tryptophan solution), which has previously been added to the soft agar:
300 mL solution B (agar)
100 mL solution A (saline solution)
8 mL solution C (glucose solution)
10 mL solution D (casein solution)
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (trp+ revertants) are counted.

PREINCUBATION TEST:
The experimental procedure is based on the method described by Yahagi et al. (Mut. Res. 48: 121-130, 1977) and Matsushima et al. (Factors modulating mutagenicity in microbial tests. In: Norpoth, K.H. and R.C. Garner, Short-Term Test Systems for Detecting Carcinogens. Springer Verlag Berlin, Heidelberg, New York, 1980):
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) are incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.

CONTROLS:
Negative control:
Parallel with each experiment with and without S9-mix, negative controls (solvent control, sterility control) were carried out for each tester strain in order to determine the spontaneous mutation rate.
Positive controls:
The following positive control substances were used to check the mutability of the bacteria and the activity of the S9-mix. With S9 mix: 2-aminoanthracene, 2-AA (2.5 µg/plate for all 4 Salmonella strains, 60 µg/plate for E. coli strain); without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine, MNNG (5 µg/plate for TA 1535 and TA100); 4-nitro-o-phenylenediamine, NOPD (10 µg/plate for TA 98), 4-nitroquinoline-1-oxide, 9-Aminoacridine, AAC (100 µg/plate for TA 1537), 4-NQO (5 µg/plate for E. coli WP2 uvrA).

TITER DETERMINATION:
The titer was determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.
In the standard plate test, 0.1 mL of the overnight cultures is diluted to 10E-6 in each case. Test tubes containing 2 mL portions of soft agar containing maximal amino acid solution (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:
0.1 mL vehicle (without and with test substance)
0.1 mL fresh bacterial culture (dilution: 10E-6)
0.5 mL S9 mix
In the preincubation test, 0.1 mL of the overnight cultures is diluted to 10E-6 in each case. 0.1 mL vehicle (with and without test substance), 0.1 mL bacterial suspension and 0.5 mL S9 mix are incubated at 37°C for about 20 minutes using a shaker. Subsequently, 2 mL of soft agar containing maximal amino acid solution for titer determination (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) is added.
After mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.

Evaluation criteria:

Assessment criteria:
The test chemical is considered positive in this assay if the following criteria are met:
A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if the number of revertants for all tester strains are within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Acceptance criteria:
Generally, the experiment is considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls is within the range of the historical negative control data for each tester strain.
- The sterility control reveales no indication of bacterial contamination.
- The positive control substances both with and without S9 mix induce a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
- The titer of viable bacteria is >= 10E+8/mL.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 2500 µg/plate in standard plate test; >= 1250 µg/plate in preincubation test

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 5000 µg/plate for both standard plate test and preincubation test

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MUTAGENICITY:
No increase in the number of his+ or trp+ revertants was observed, neither in the standard plate test nor in the preincubation test, with or without the addition ofS9 mix.
The results of the negative and positive controls were as expected and confirmed the validity and sensitivity of the test system used.

SOLUBILITY:
Precipitation of the test substance was found from 100 μg/plate onward without S9 mix and from about 250 μg/plate onward with S9 mix.

TOXICITY:
In the standard plate test, a bacteriotoxic effect was observed with or without S9 mix from 2500 μg/plate onward.
In the preincubation assay bacteriotoxicity was observed with or without S9 mix from 1250 μg/plate onward for the tested Salmonella strains and from 5000 μg/plate onward for the tested E. coli strain.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: in standard plate test and preincubation test

Any other information on results incl. tables

Table 1: Maximum revertants/plate and corresponding test concentrations in the standard plate test:
Strain	Tested compound	Maximum revertants/plate [corresponding dose unit in µg/plate]
		without S9-mix	with S9-mix
S. typhimurium TA1535	DMSO	16 ± 1	14 ± 2
	Test substance	19 ± 2 [500]	18 ± 2 [20]
	Positive Control	594 ± 38 [5; MNNG]	133 ± 18 [2.5; 2-AA]
S. typhimurium TA100	DMSO	103 ± 6	103 ± 11
	Test substance	104 ± 5 [500]	103 ± 8 [100]
	Positive Control	668 ± 32 [5; MNNG]	837 ± 98 [2.5; 2-AA]
S. typhimurium TA98	DMSO	25 ± 3	36 ± 4
	Test substance	25 ± 6 [500]	35 ± 5 [100]
	Positive Control	497 ± 65 [10; NOPD]	610 ± 18 [2.5; 2-AA]
S. typhimurium TA1537	DMSO	8 ± 2	8 ± 3
	Test substance	10 ± 5 [20]	10 ± 3 [20]
	Positive Control	367 ± 36 [100; AAC]	119 ± 29 [2.5; 2-AA]
E. coli	DMSO	38 ± 7	39 ± 6
WP2 uvrA	Test substance	46 ± 12 [500]	43 ± 10 [500]
	Positive Control	594 ± 39 [5; 4-NQO]	254 ± 25 [60; 2-AA]
2-AA = 2-aminoanthracene
MNNG = N-methyl-N'-nitro-N-nitrosoguanidine
NOPD = 4-nitro-o-phenylenediamine
AAC = 9-aminoacridine
4-NQO = 4-nitroquinoline-N-oxide

Table 2: Maximum revertants/plate and corresponding test concentrations in the preincubation test:
Strain	Tested compound	Maximum revertants/plate [corresponding dose unit in µg/plate]
		without S9-mix	with S9-mix
S. typhimurium TA1535	DMSO	14 ± 1	14 ± 2
	Test substance	14 ± 4 [500]	14 ± 5 [10]
	Positive Control	1012 ± 43 [5; MNNG]	117 ± 37 [2.5; 2-AA]
S. typhimurium TA100	DMSO	104 ± 3	101 ± 6
	Test substance	106 ± 6 [2500]	107 ± 12 [10]
	Positive Control	1179 ± 136 [5; MNNG]	741 ± 20 [2.5; 2-AA]
S. typhimurium TA98	DMSO	28 ± 2	33 ± 4
	Test substance	27 ± 1 [20]	31 ± 4 [50]
	Positive Control	415 ± 15 [10; NOPD]	557 ± 15 [2.5; 2-AA]
S. typhimurium TA1537	DMSO	9 ± 3	8 ± 2
	Test substance	9 ± 2 [20]	9 ± 4 [50]
	Positive Control	432 ± 12 [100; AAC]	113 ± 6 [2.5; 2-AA]
E. coli	DMSO	30 ± 3	29 ± 2
WP2 uvrA	Test substance	28 ± 2 [500]	38 ± 9 [500]
	Positive Control	791 ± 24 [5; 4-NQO]	197 ± 18 [60; 2-AA]
2-AA = 2-aminoanthracene
MNNG = N-methyl-N'-nitro-N-nitrosoguanidine
NOPD = 4-nitro-o-phenylenediamine
AAC = 9-aminoacridine
4-NQO = 4-nitroquinoline-N-oxide