[1,1'-Biphenyl]-2-carboxylic acid, 4'-(bromomethyl)-, methyl 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

Study period:

15 Septiembre 2013 - 19 December 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test method according to OECD 471. GLP study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

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:

methyl 4'-(bromomethyl)-[1,1'-biphenyl]-2-carboxylate

EC Number:

601-325-6

Cas Number:

114772-38-2

Molecular formula:

C15H13BrO2

IUPAC Name:

methyl 4'-(bromomethyl)-[1,1'-biphenyl]-2-carboxylate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material: Methyl 2-[4-(bromomethyl)phenyl]benzoate
- Physical state: white powder
- Analytical purity: 100.7%
- Lot/batch No.: M13552C

Method

Target gene:

Histidine-requiring gene in Salmonella typhimurium and Tryptophan-requiring gene in the strain of Escherichia coli.

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:

S9 mix from rat liver

Test concentrations with justification for top dose:

Initial Mutation Test: 5000; 2500; 1000; 316; 100; 31.6 and 10 µg test item/plate
Confirmatory Mutation Test: 5000, 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 µg test item/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of the test item was examined using Distilled water, Dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF). The test item was insoluble in Distilled water. The formulation at 100 mg/mL concentration using DMSO or DMF as vehicle was suitable for the test. Due to the biocompatibility to the test system, DMSO was selected as vehicle of the study.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and distilled water

Controls 2

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100, TA1535 -S9 (2 µg/plate)

Controls 3

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene-diamine

Remarks:

TA98, -S9 (4 µg/plate)

Controls 4

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537, -S9 (50 µg/plate)

Controls 5

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

WP2 uvrA, -S9 (2 µL/plate)

Controls 6

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

all Salmonella strains, +S9 (2 µg/plate) and WP2 uvrA, +S9 (50 µg/plate)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Plate incorporation (initial mutation test and confirmatory test):
Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C). This solution was mixed and poured on the surface of minimal agar plates. After preparation, the plates were incubated at 37°C for 48 hours.

NUMBER OF REPLICATIONS: 3 replicates per dose and controls.

EVALUATION OF EXPERIMENTAL DATA:
The colony numbers were determined by manual counting.
Visual examination of the plates was performed, precipitation or signs of growth inhibition (if any) were recorded and reported.
The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated.

Evaluation criteria:

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.
Criteria for a Negative Response:
A test article was considered non-mutagenic if it produced neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

(TA98, TA100, TA1535 and WP2 uvrA, with and without metabolic activation)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(TA98, TA100 and TA1537, with and without metabolic activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Sporadically, slightly lower revertant counts compared to the solvent control were observed in the main tests at some non-cytotoxic concentrations. However, the mean number of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Inhibitory, cytotoxic effect of the test item (absent/reduced/slightly reduced background lawn development and / or reduced number of revertant colonies, in some cases pinpoint colonies were also detected) were observed in the Initial Mutation Test in Salmonella typhimurium TA98 strain at 5000 µg/plate without metabolic activation; in Salmonella typhimurium TA 100 strain at 5000, 1581 and 500 µg/plate concentrations with metabolic activation and in Salmonella typhimurium TA1537 strain at 5000, 1581 and 500 µg/plate concentrations with and without metabolic activation.
Similar inhibitory, cytotoxic effect of the test item were observed in the Confirmatory Mutation Test in Salmonella typhimurium TA 98 strain at 5000 and 1581 µg/plate without metabolic activation; in Salmonella typhimurium TA100 and TA1537 strains at 5000, 1581 and 500 µg/plate concentrations without metabolic activation and in Salmonella typhimurium TA100 and TA1537 strains at 5000 and 1581 µg/plate concentrations with metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Initial Mutation Test

Concentrations (µg/plate)	Mean values of revertants /Mutation factor (MF)	Salmonella typhimurium tester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2 uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
5000	Mean	482.7	442.7	101.3	242.0	100.7	179.0	0.0	0.3	120.3	121.0
	MF	20.39	15.81	1.03	2.70	7.02	23.35	0.00	0.05	3.11	2.81
1581	Mean	456.7	476.7	124.3	172.0	73.7	124.0	2.0	6.0	101.0	105.7
	MF	19.30	17.02	1.27	1.92	5.14	16.17	0.40	0.86	2.61	2.46
500	Mean	237.3	394.0	174.0	260.7	41.3	47.0	2.7	8.7	58.3	85.7
	MF	10.03	14.07	1.78	2.91	2.88	6.13	0.53	1.24	1.51	1.99
158.1	Mean	206.0	177.3	238.0	252.3	24.3	19.7	8.3	15.7	41.0	51.0
	MF	8.70	6.33	2.43	2.81	1.70	2.57	1.67	2.24	1.06	1.19
50	Mean	87.7	61.7	197.7	135.7	17.0	13.0	13.7	15.0	32.3	41.7
	MF	3.70	2.20	2.02	1.51	1.19	1.70	2.73	2.14	0.84	0.97
15.81	Mean	49.7	33.3	130.7	89.0	15.0	9.7	15.0	9.3	39.0	41.3
	MF	2.10	1.19	1.33	0.99	1.05	1.26	3.00	1.33	1.01	0.96
5	Mean	31.0	30.3	113.7	92.7	22.0	8.7	5.0	10.7	46.7	44.0
	MF	1.31	1.08	1.16	1.03	1.53	1.13	1.00	1.52	1.21	1.02
1.581	Mean	23.3	29.0	107.3	91.3	11.3	8.7	7.3	11.0	42.3	36.7
	MF	0.99	1.04	1.10	1.02	0.79	1.13	1.47	1.57	1.09	0.85

 Confirmatory Mutation Test

Concentrations (µg/plate)	Mean values of revertants /Mutation factor (MF)	Salmonella typhimurium tester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2 uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
5000	Mean	176.3	313.3	55.0	73.7	123.0	171.3	3.7	3.7	117.0	140.7
	MF	5.24	8.62	0.69	0.88	10.85	16.06	0.69	0.50	3.31	2.78
1581	Mean	170.7	448.0	77.7	93.3	80.0	116.0	2.3	8.0	106.3	132.0
	MF	5.07	12.33	0.98	1.12	7.06	10.88	0.44	1.09	3.01	2.61
500	Mean	275.7	296.7	131.0	147.0	33.3	49.3	5.3	11.0	91.7	154.7
	MF	8.19	8.17	1.65	1.76	2.94	4.63	1.00	1.50	2.59	3.05
158.1	Mean	235.7	194.7	190.3	219.7	27.7	20.3	8.3	17.0	70.3	78.0
	MF	7.00	5.36	2.40	2.63	2.44	1.91	1.56	2.32	1.99	1.54
50	Mean	118.0	77.7	160.7	140.3	20.3	14.0	5.7	6.3	38.0	46.7
	MF	3.50	2.14	2.03	1.68	1.79	1.31	1.06	0.86	1.08	0.92
15.81	Mean	64.0	35.0	128.0	93.3	20.3	10.3	4.3	5.7	32.3	47.0
	MF	1.90	0.96	1.61	1.12	1.79	0.97	0.81	0.77	0.92	0.93
5	Mean	52.0	32.7	105.7	81.0	28.7	6.0	3.0	6.7	35.7	37.7
	MF	1.54	0.90	1.33	0.97	2.53	0.56	0.56	0.91	1.01	0.74
1.581	Mean	31.0	33.0	89.7	94.0	12.7	10.7	5.3	5.7	37.3	46.0
	MF	0.92	0.91	1.13	1.12	1.12	1.00	1.00	0.77	1.06	0.91
0.5	Mean	26.3	34.7	98.7	96.3	14.7	13.0	3.7	6.7	38.0	47.7
	MF	0.78	0.95	1.24	1.15	1.29	1.22	0.69	0.91	1.08	0.94

In the initial mutation test, a clear positive effect of the test item was obtained in Salmonella typhimurium TA98, TA100, TA1535 and Escherichia coli WP2 uvrA bacterial strains with and without metabolic activation and in Salmonella typhimurium TA1537 strain without metabolic activation. Increased revertant counts were detected in Salmonella typhimurium TA1537 strain with metabolic activation in some cases.

The dose dependent substantial increases over the threshold limits were confirmed in the Confirmatory Mutation Test using the same experimental conditions. All the results were confirmed except for the Salmonella typhimurium TA1537 strain without metabolic activation.

Inhibitory, cytotoxic effect of the test item were observed in the Initial Mutation Test in Salmonella typhimurium TA98 strain at 5000 µg/plate without metabolic activation; in Salmonella typhimurium TA100 strain at 5000, 1581 and 500 µg/plate concentrations without metabolic concentration; in this strain at 5000 and 1581 µg/plate concentrations with metabolic activation and in Salmonella typhimurium TA1537 strain st 5000, 1581 and 500 µg/plate concentrations with and without metabolic activation.

Similar inhibitory, cytotoxic effect of the test item were observed in the Confirmatory Mutation Test in Salmonella typhimurium TA98 strain at 5000 and 1581 µg/plate without metabolic activation; in Salmonella typhimurium TA100 and TA 1537 strains at 5000, 1581 and 500µg/plate concentrations without metabolic activation and in Salmonella typhimurium TA100 and TA1537 strains at 5000 and 1581µg/plate concentrations with metabolic activation.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive (with and without metabolic activation)

The test item had mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD Guideline 471 (GLP study). The experiments were carried out using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA in the presence and absence of metabolic activation (S9 fraction prepared from the livers of rats). Based on the results of the Solubility Test, the test item was dissolved in DFMO. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the main tests were as follows: Initial Mutation Test: 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 µg test item/plate and the test item concentrations. Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5µg test item/plate. In the Initial Mutation Test and Confirmatory Mutation Test and Confirmatory Mutation Tests (using the plate incorporation method), a clear, reproducible positive effect was obtained in four examined bacterial strains (Salmonella typhimurium TA98, TA100, TA1535 and Escherichia coli WP2 uvrA) with and without metabolic activation as the observed revertant colony numbers were above the respective biological threshold value and dose-related trends were also observed.