Methyl 2-benzoylbenzoate

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:

2007 -11-29 till 2007-12-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

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:

Methyl 2-benzoylbenzoate

EC Number:

210-112-3

EC Name:

Methyl 2-benzoylbenzoate

Cas Number:

606-28-0

Molecular formula:

C15H12O3

IUPAC Name:

methyl 2-benzoylbenzoate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identity: GENOCURE* MBB
Chemical Name: Methyl 2-benzoylbenzoate
CAS No.: 606-28-0
Batch No.: G07001
Aggregate state at room temperature: solid
Colour: Off white, light yellow
Molecular weight:240.26 g/mol
Purity: > 99 %
Solubility: Soluble in water <1 g/L
Stability in solvent:Stable for at least 30 days at THF and acetone
Storage: At room temperature
Expiration Date: June 30, 2008

Specific details on test material used for the study:

Identity: GENOCURE* MBB
Chemical Name: Methyl 2-benzoylbenzoate
CAS No.: 606-28-0
Batch No.: G07001
Aggregate state: Solid
Colour: off-white, light yellow
Molecular weight: 240.26 g/mol
Purity: > 99 %
Solubility: soluble in water <1 g/L
Stability in solvent: stable for at least 30 days at THF and acetone
Storage: at room temperature
Expiration Date: June 30, 2008

Method

Species / strain

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I
33; 100; 333; 1000; 2500; and 5000 µg/plate / experiment II

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dry THF
- Justification for choice of solvent/vehicle: chosen because of its solubility properties

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation and preincubation method


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation:
The test item precipitated in the overlay agar in the test tubes from 2500 µg/plate up to 5000 µg/plate in experiment I and at 5000 µg/plate in experiment II. The undissolved particles had no influence on the data recording.

COMPARISON WITH HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY:

The plates incubated with the test item showed reduced background growth at the following concentrations (µg/plate):
Strain Experiment I Experiment II
without S9 mix with S9 mix without S9 mix with S9 mix
TA 1535 / / / 2500 - 5000
TA 1537 / / / 1000 - 5000
TA 98 / / / /
TA 100 / / 1000 - 5000 333 - 5000
WP2 uvrA / / / /
/ = no reduced background growth
No toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), occurred in the test groups with and without metabol¬ic activation. Only in strain TA 1537 with metabolic activation a reduction in the number of revertants (below the induction factor of 0.5) was observed from 2500 µg/plate up to 5000 µg/plate in experiment I and at 1000 µg/plate in experiment II.

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

1.1   Summary of Results Pre-Experiment/Experiment I

Study Name: 1142601	Study Code: RCC-CCR 1142601
Experiment: 1142601 VV Plate	Date Plated: 29/11/2007
Assay Conditions:	Date Counted: 05/12/2007

 

Metabolic Activation	Test Group	Dose Level (µg/plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	THF			11 ± 1^B M	14 ± 4	33 ± 12	128 ± 5	37 ± 5
	Untreated			14 ± 1^B M	12 ± 5	23 ± 3	135 ± 13	36 ± 5
	GENOCURE*	3 µg		13 ± 3^B M	12 ± 6	37 ± 4	133 ± 8	42 ± 5
	MBB	10 µg		11 ± 1^B M	15 ± 6	30 ± 10	133 ± 17	39 ± 4
		33 µg		13 ± 3^B M	17 ± 5	28 ± 4	126 ± 3	41 ± 5
		100 µg		10 ± 2^B M	13 ± 6	39 ± 4	129 ± 6	41 ± 6
		333 µg		11 ± 1^B M	15 ± 2	25 ± 10	120 ± 18	40 ± 6
		1000 µg		11 ± 2^B M	15 ± 2	25 ± 4	115 ± 18	38 ± 5
		2500 µg		9 ± 1^B M	11 ± 4	30 ± 4	115 ± 9	40 ± 3
		5000 µg		11 ± 2^B M	11 ± 4	29 ± 9	128 ± 14	40 ± 4
	NaN3	10 µg		1880 ± 85			1868 ± 118
	4-NOPD	10 µg				502 ± 33
	4-NOPD	50 µg			103 ± 10
	MMS	3.0 µL						1163 ± 27
With Activation	THF			15 ± 1^B M	24 ± 7	39 ± 4	128 ± 12	55 ± 15
	Untreated			12 ± 1^B M	18 ± 4	33 ± 9	126 ± 13	54 ± 12
	GENOCURE*	3 µg		12 ± 1^B M	21 ± 7	36 ± 7	117 ± 7	52 ± 13
	MBB	10 µg		12 ± 2^B M	24 ± 8	36 ± 6	122 ± 12	64 ± 14
		33 µg		15 ± 1^B M	25 ± 3	33 ± 3	131 ± 16	64 ± 7
		100 µg		12 ± 2^B M	17 ± 7	39 ± 5	143 ± 7	57 ± 2
		333 µg		11 ± 3^B M	26 ± 7	39 ± 8	122 ± 17	59 ± 10
		1000 µg		8 ± 2^B M	17 ± 3	36 ± 5	99 ± 9	50 ± 10
		2500 µg		8 ± 2^B M	11 ± 4	35 ± 7	107 ± 2	52 ± 7
		5000 µg		8 ± 3^B M	9 ± 2	38 ± 3	95 ± 11	48 ± 8
	2-AA	2.5 µg		366 ± 30	134 ± 5	905 ± 67	1350 ± 32
	2-AA	10.0 µg						315 ± 33

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	B M	Extensive bacterial growth Manual count

 

1.2   Summary of Results Experiment II

 

Study Name: 1142601	Study Code: RCC-CCR 1142601
Experiment: 1142601 HV2 Pre	Date Plated: 06/12/2007
Assay Conditions:	Date Counted: 11/12/2007

 

Metabolic Activation	Test Group	Dose Level (µg/plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	THF			14 ± 3^B M	11 ± 4	30 ± 11	119 ± 5	45 ± 2
	Untreated			20 ± 1^B M	11 ± 6	26 ± 8	131 ± 10	50 ± 13
	GENOCURE*	33 µg		12 ± 4^B M	11 ± 6	22 ± 7	109 ± 19	46 ± 11
	MBB	100 µg		12 ± 3^B M	9 ± 3	22 ± 11	122 ± 16	30 ± 9
		333 µg		11 ± 1^B M	12 ± 3	29 ± 11	115 ± 7	41 ± 13
		1000 µg		11 ± 2^B M	8 ± 1	20 ± 7	69 ± 3^R	45 ± 10
		2500 µg		11 ± 4^B M	12 ± 5	21 ± 12	67 ± 4^R	36 ± 9
		5000 µg		14 ± 3^B M	12 ± 3	23 ± 5	65 ± 20^R M	41 ± 8
	NaN3	10 µg		2042 ± 97			2060 ± 25
	4-NOPD	10 µg				607 ± 9
	4-NOPD	50 µg			127 ± 21
	MMS	3.0 µL						281 ± 21
With Activation	THF			16 ± 4^B M	23 ± 4	36 ± 5	135 ± 15	64 ± 8
	Untreated			16 ± 3^B M	19 ± 3	38 ± 2	150 ± 12	57 ± 7
	GENOCURE*	33 µg		18 ± 4^B M	18 ± 5	40 ± 5	144 ± 9	58 ± 13
	MBB	100 µg		13 ± 4^B M	18 ± 5	35 ± 6	135 ± 13	60 ± 12
		333 µg		12 ± 2^B M	16 ± 4	34 ± 1	99 ± 14^R	56 ± 1
		1000 µg		12 ± 3^B M	9 ± 3^R	32 ± 5	78 ± 1^R	56 ± 4
		2500 µg		8 ± 3^B M R	12 ± 3^R	27 ± 7	104 ± 10^R	47 ± 3
		5000 µg		8 ± 2^B M R	12 ± 3^R	23 ± 5	88 ± 18^R	56 ± 4
	2-AA	2.5 µg		129 ± 20	120 ± 6	797 ± 72	1052 ± 28
	2-AA	10.0 µg						334 ± 17

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	R M B	Reduced background growth Manual count Extensive bacterial growth

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without S9 MIx

In conclusion, it can be stated that during the described mutage¬nicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

This study was performed to investigate the potential of GENOCURE* MBB to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:         3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                                   33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal back­ground growth up to 5000 µg/plate with and without metabolic activation in experiment I. Reduced background growth was observed in experiment II in few strains.

No toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), occurred in the test groups with and without metabol­ic activation. Only in strain TA 1537 with metabolic activation a reduction in the number of revertants (below the induction factor of 0.5) was observed from 2500 µg/plate up to 5000 µg/plate in experiment I and at 1000 µg/plate in experiment II.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with GENOCURE* MBB at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher muta­tion rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct in­crease of induced revertant colonies.