Methyl 2-benzoylbenzoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is non-mutagenic in 2 in vitro mutagenicity assays (Ames test, in vitro gene mutation assay) and slightly positive in the in vitro chromosome aberration assay, only in the presence of S-9 mix.

Link to relevant study records

Reference

Reference 1

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

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

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

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

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.

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'.

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

 

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-11-06 until 2007-12-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

first experiment: 4 hours treatment with and without metabolic activation
second experiment: 24 hours treatment without metabolic activation, 4 hours treatment with metaoblic activation

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

Identity: GENOCURE* MBB
Chemical Name: Methyl 2-benzoylbenzoate
Batch No.: G07001
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

Target gene:

Thymidine Kinase Locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

other: Clone 3.7.2C

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

Experiment I:
without S9 mix: 18.8; 37.5; 75.0; 150.0; 225.0; 300.0 µg/mL
with S9 mix: 37.5; 75.0; 150.0; 300.0; 600.0; 1200.0 µg/mL
Experiment II:
without S9 mix: 37.5; 75.0; 150.0; 300.0; 600.0; 1200.0 µg/mL
Following the expression phase of 48 hours the cultures of the lowest concentration (printed in bold letters) in the first experiment with S9 mix were not continued since a minimum of only four concentrations is required by the guidelines. The cultures at 225 and 300 µg/mL in experiment I without S9 mix were not continued due to exceedingly severe toxic effects. The cultures of the maximum concentration of 1200 µg/mL in the second experiment (without S9 mix) were not continued for the same reason.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: solubility properties

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours with and without metabolic activation in experiment 1, 24 hours without metaoblic activation in experiment and 4 hours with metabolic activation in experiment 2
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 to 15 days

SELECTION AGENT (mutation assays): RPMI 1640 medium by addition of 5 µg/mL TFT

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1,5 x 10 exp. 6 cells

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10 exp. 6 cells above the corresponding solvent control or negative control, respectively.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in negative
and/or vehicle con¬trols and the mutation rates of all negative and/or vehicle controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth, and the cloning efficiency 1 is less than 10 % of the vehicle control
unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used
to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and
dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.

Statistics:

Linear regression analysis (least squares) using SYSTAT (R) 11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA)

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (solvent control: pH 7.4, max. conc. of 2400 µg/mL: pH 7.4) measured in the pre-experiment
- Effects of osmolality: Not increased (solvent control: 346 mOsm, max. conc. of 2400 µg/mL: 325 mOsm) measured in the pre-experiment
- Evaporation from medium: Not examined
- Water solubility: Not indicated
- Precipitation:
The test medium was checked for precipitation visible to the naked eye at the end of each treatment period (4 or 24 hours) just before the test item was removed. In experiment I precipitation was determined only at the maximum concentration of 1200 µg/mL in the presence of metabolic activation. In experiment II precipitation was noted at 300 µg/mL and above.

- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
A pre-test was performed in order to determine the concentration range of the mutagenicity experiments. Both, pH value and osmolarity were determined at the maximal concentration of the test item and in the solvent control without metabolic activation.
1x107 cells were exposed to each concentration of the test item for 4 and 24 hours without and 4 hours with metabolic activation. During the 4 h treatment period the serum concentration was reduced from 15 % to 3 %. Following treatment the cells were washed twice by centrifugation (425 g, 10 min) and resuspended in "saline G". Subsequently the cells were resuspended in 30 mL complete culture medium for a 2-day growth period. The cell density was determined immediately after treatment and at each day of the growth period and adjusted to 3x105 cells/mL, if necessary. The relative suspension growth (RSG) of the treated cell cultures was calculated at the end of the growth period according to the method of Clive and Spector.
The highest concentration used in the pre-test was chosen with regard to the guidelines cited. Test item concentrations between 18.8 and 2400 µg/mL were used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation.
In this pre-experiment strong toxic effects were observed at 300 µg/mL and above in the absence and at 1200 µg/mL and above in the presence of metabolic activation at pulse treatment. Following continuous treatment toxic effects occurred at the maximum concentration of 2400 µg/mL.
The test medium was checked for precipitation at the end of each treatment period (4 or 24 hours) before the test item was removed. Precipitation of the test item was observed at 300 µg/mL and above without and at 600 µg/mL and above with metabolic activation following 4 hour treatment. After 24 hour treatment precipitation occurred at 600 µg/mL and above.
The test item did not alter the osmolarity and pH-value even at the maximum concentration.
According to the results of the pre-experiment the dose range of the main experiments was chosen. The concentration range of the first experiment without metabolic activation was limited to 300 µg/mL by toxic effects. In the second experiment and in the first experiment with metabolic activation the dose range was limited to 1200 µg/mL by precipitation of the test item. The individual concentrations were generally spaced by a factor of 2. A narrower spacing was used at the two highest concentrations of the first experiment without metabolic activation to cover the toxic range more closely. To overcome problems with possible deviations in toxicity both main experiments were started with more than four concentrations.


COMPARISON WITH HISTORICAL CONTROL DATA: Complies

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant toxic effects indicated by a relative cloning efficiency 1 and/or a relative total growth of less than 50 % were observed in the first experiment without metabolic activation at 150 µg/mL (4 hours of treatment). In the second experiment (24 h treatment) toxic effects as described above occurred in both parallel cultures at 1200 µg/mL. At the second precipitating concentration of 600 µg/mL toxic effects were noted in culture I but not in culture II. Such irreproducible effects are common in the precipitating concentration range. At the end of treatment the test item is removed from the cells by centrifugation. A precipitate contaminates the cell pellet and is carried over to the next steps of the assay leading to non-defined treatment intervals with arbitrary amounts of test item. The data generated in culture I of the second experiment at 600 µg/mL are not considered valid since both parameters of toxicity (survival and RTG) remained far below the lower threshold of 10 %.

Remarks on result:

other: strain/cell type: in vitro gene mutation assay with L5178Y cells

Remarks:

Migrated from field 'Test system'.

Summary Table

			relative	relative	mutant		relative	relative	mutant
	conc. µg	S9	cloning	total	colonies/		cloning	total	colonies/
	per mL	mix	efficiency 1	growth	10^6cells	threshold	efficiency 1	growth	10^6cells	threshold
Column	1	2	3	4	5	6	7	8	9	10
Experiment I / 4 h treatment			culture I				culture II
Neg. control with medium		-	100.0	100.0	160		100.0	100.0	128
Solv. control with THF		-	100.0	100.0	107	233	100.0	100.0	146	272
Pos. control with MMS	19.5	-	55.4	44.9	319	233	53.6	36.6	350	272
Test item	18.8	-	104.8	78.5	157	233	90.1	124.5	93	272
Test item	37.5	-	90.0	96.2	128	233	80.2	101.4	124	272
Test item	75.0	-	87.4	71.4	164	233	72.6	125.6	79	272
Test item	150.0	-	29.6	19.9	219	233	37.1	33.0	111	272
Test item	225.0	-	1.8	culture was not continued^#			1.8	culture was not continued^#
Test item	300.0	-	0.4	culture was not continued^#			0.7	culture was not continued^#
Neg. control with medium		+	100.0	100.0	168		100.0	100.0	127
Solv. control with THF		+	100.0	100.0	167	293	100.0	100.0	141	267
Pos. control with CPA	3.0	+	68.3	53.4	303	293	110.4	44.2	258	267
Pos. control with CPA	4.5	+	33.8	28.5	320	293	63.4	24.8	406	267
Test item	37.5	+	98.3	culture was not continued^##			103.1	culture was not continued^##
Test item	75.0	+	129.1	90.3	161	293	72.1	91.2	150	267
Test item	150.0	+	91.9	100.2	132	293	69.4	109.9	150	267
Test item	300.0	+	81.0	105.2	162	293	61.4	80.0	126	267
Test item	600.0	+	95.0	71.2	205	293	57.5	86.2	149	267
Test item	1200 (p)	+	81.0	85.0	177	293	50.8	100.2	150	267
Experiment II / 24 h treatment			culture I				culture II
Neg. control with medium		-	100.0	100.0	119		100.0	100.0	81
Solv. control with THF		-	100.0	100.0	117	243	100.0	100.0	88	214
Pos. control with MMS	13.0	-	19.2	16.4	634	243	23.4	11.0	587	214
Test item	37.5	-	98.5	141.0	73	243	103.5	51.1	94	214
Test item	75.0	-	126.3	126.8	129	243	105.3	81.4	121	214
Test item	150.0	-	104.6	129.5	105	243	100.0	90.8	133	214
Test item	300 (p)	-	49.2	87.8	57	243	76.6	82.0	156	214
Test item	600 (p)	-	2.8	1.5	164	243	69.1	59.5	131	214
Test item	1200 (p)	-	0.4	culture was not continued^#			2.1	culture was not continued^#

#    culture was not continued due to strong toxic effects
##  culture was not continued since only a minimum of four concentrations is required by the guidelines

(p)   precipitation visible to the unaided eye

the bold printed values are not considered valid due to exceedingly severe cytotoxic effects

 

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Executive summary:

The study was performed to investigate the potential of GENOCURE* MBB to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second main experiment was solely performed without metabolic activation using a treatment time of 24 h. In the main experiments the test item was evaluated at the following concentrations:

Experiment I

without S9 mix:                                                     18.8; 37.5; 75; and 150 µg/mL
with S9 mix:                                                         75; 150; 300; 600; 1200 µg/mL

Experiment II

without S9 mix:                                              37.5; 75; 150; 300; and 600 µg/mL

The test medium was checked for precipitation visible to the naked eye at the end of each treatment period (4 or 24 hours) just before the test item was removed. In experiment I precipitation was determined only at the maximum concentration of 1200 µg/mL in the presence of metabolic activation. In experiment II precipitation was noted at 300 µg/mL and above. Relevant toxic effects indicated by a relative cloning efficiency 1 and/or a relative total growth of less than 50 % were observed in the first experiment without metabolic activation at 150 µg/mL (4 hours of treatment). In the second experiment (24 h treatment) toxic effects as described above occurred in both parallel cultures at 1200 µg/mL. At the second precipitating concentration of 600 µg/mL toxic effects were noted in culture I but not in culture II. Such irreproducible effects are common in the precipitating concentration range. At the end of treatment the test item is removed from the cells by centrifugation. A precipitate contaminates the cell pellet and is carried over to the next steps of the assay leading to non-defined treatment intervals with arbitrary amounts of test item. The data generated in culture I of the second experiment at 600 µg/mL are not considered valid since both parameters of toxicity (survival and RTG) remained far below the lower threshold of 10 %.

In both experiments the number of mutant colonies/10⁶cells generally remained well within the range of the historical solvent control data in the presence and absence of metabolic activation. Two moderate increases of mutant colonies/10⁶cells exceeding the historical range occurred in the first experiment at 150 µg/mL without metabolic activation (219 mutant colonies/10⁶cells) and at 600 µg/mL with metabolic activation (205 mutant colonies/10⁶cells). However, the moderate increase described above was judged as biologically irrelevant for the following reasons. The threshold of 126 above the mutation frequency of the corresponding solvent control was not reached and the mean values of the mutation frequency of both parallel cultures (219 plus 111 equal to a mean of 165 and 205 plus 149 equal to a mean of 177) remained within the historical control range.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency using SYSTAT(R) statistics software. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the first culture of experiment I without metabolic activation. In the second experiment a significant dose dependent trend was observed in culture II. Since the mutation frequencies at these test points did not exceed the threshold as indicated above and no significant increase occurred in the parallel cultures under identical conditions, the statistical results are considered as biologically irrelevant fluctuations.

In this study the range of the negative and solvent controls was from 88 up to 168 mutant colonies per 10⁶cells; the range of the groups treated with the test item was from 57 up to 219 mutant colonies per 10⁶cells. MMS (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and CPA (3.0 and 4.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies at at least one of the concentrations of the controls.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Identity: GENOCURE* MBB
Chemical Name: Methyl 2-benzoylbenzoate
Batch No.: G07001
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

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: minimal essential medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital / beta-naphtoflavone induced rat liver S9

Test concentrations with justification for top dose:

With metabolic activation:
Experiment I: 75.3, 150.6, 301.3, 1205.0 µg/mL

Without metabolic activation:
Experiment I: 37.7, 75.3, 150.6 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: THF
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

According to the OECD Guideline only one experiment was performed, since the test item was considered to be mutagenic after 4 hours treatment. The chromosomes were prepared 18 hours after start of treatment with the test item. The exposure period was 4 hours with and without metabolic activation. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium (minimal essential medium)

DURATION
- Exposure duration: 4 hours (+/- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 18 hours


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: about 1.5


NUMBER OF CELLS EVALUATED: at least 100 per culture


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and cell numbers


OTHER EXAMINATIONS:
- Determination of polyploidy: 500 per culture
- Determination of endoreplication: 500 per culture

Evaluation criteria:

Evaluation of the cultures was performed according to the OECD Guideline using NIKON microscopes with 100x objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) and relative cell numbers were determined.
In addition, the number of polyploid cells in 500 metaphases per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype). Additionally the number of endomitotic cells scored at the evaluation of polyploid cells was noticed and reported (% endomitotic metaphases).

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item GENOCURE* MBB, dissolved in tetrahydrofurane (THF), was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in the absence and the presence of metabolic activation by S9 mix.
According to the OECD Guideline only one experiment was performed, since the test item was considered to be mutagenic after 4 hours treatment. The chromosomes were prepared 18 hours after start of treatment with the test item. The exposure period was 4 hours with and without metabolic activation.
In each experimental group two parallel cultures were set up. Per culture at least 100 metaphases were scored for structural chromosome aberrations.
In a range finding pre-test on toxicity cell numbers were scored 24 hours after start of treatment as an indicator for cytotoxicity. Concentrations between 18.8 and 2410 µg/mL were applied. Clear toxic effects were observed after 4 hours treatment with 301.3 µg/mL and above in the absence of S9 mix and with 2410 µg/mL in the presence of S9 mix. Precipitation of the test item in culture medium was observed after 4 hours treatment with 301.3 µg/mL and above in the absence of S9 mix and 602.5 µg/mL and above in the presence of S9 mix. No relevant influence of the test item on the pH value or osmolarity was observed (solvent control 425 mOsm, pH 7.1 versus 358 mOsm and pH 7.2 at 2410 µg/mL).
In the main Experiment precipitation of the test item in culture medium was observed with 301.3 µg/mL and above in the absence of S9 mix and with 602.5 µg/mL and above in the presence of S9 mix.
No toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed after treatment up to the highest evaluated test item concentrations. However, concentrations showing clearly reduced cell numbers were not evaluable for cytogenetic damage.
In the absence of S9 mix no statistically significant and biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (1.0 - 2.0 % aberrant cells, excluding gaps) compared to the solvent control value (0.5 % aberrant cells, excluding gaps) were clearly within the range of the laboratory’s historical control data range (0.0 - 4.0 % aberrant cells, excluding gaps).
In contrast in the presence of S9 mix two statistically significant and biologically relevant increases in the number of cells carrying structural chromosome aberrations (4.3 % and 5.3 %, respectively) were observed after treatment with 150.6 and 1205 µg/mL. These values clearly exceeded the laboratory’s historical control data range (0.0 – 4.0 % aberrant cells, excluding gaps). Therefore, these observations have to be regarded as biologically relevant.

No biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.7 - 3.7 %) as compared to the rates of the solvent controls (2.2 - 2.6 %). In the presence of S9 mix the frequency of endoreduplications was statistically significant increased after treatment with 301.3 µg/mL (0.4 %).

Either EMS (900 µg/mL) or CPA (1.4 µg/mL) were used as positive controls and showed distinct increases in the number of cells with structural chromosome aberrations.

Remarks on result:

other: strain/cell type: V79 cells

Remarks:

Migrated from field 'Test system'.

Summary of results of the chromosome aberration study with GENOCURE* MBB

Preparation	Test item	Polyploid	Endomitotic	Cell numbers	Mitotic indices	Aberrant cells
interval	concentration	Cells	Cells	in %	in %	in %
	in µg/mL	In %	In %	of control	of control	incl. gaps*	excl. gaps*	with exchanges
Exposure period 4 hrs without S9 mix
18 hrs	Solvent control^1	2.2	0.0	100.0	100.0	0.5	0.5	0.0
	Positive control^2	1.8	0.0	n.t.	86.2	16.5	15.5^S	6.5
	37.7	1.7	0.0	82.4	108.9	1.5	1.5	0.5
	75.3	2.2	0.0	78.6	117.8	1.0	1.0	0.5
	150.6	3.5	0.0	63.2	122.7	2.0	2.0	0.5
Exposure period 4 hrs with S9 mix
18 hrs	Solvent control^1	2.6	0.0	100.0	100.0	1.5	1.0	0.0
	Positive control^3	2.7	0.0	n.t.	112.9	10.0	10.0^S	1.5
	75.3^#	3.3	0.1	106.5	109.4	3.8	3.0	1.3
	150.6^#	3.7	0.2	94.3	118.9	4.5	4.3^S	1.3
	301.3^#	3.0	0.4^ES	96.4	120.2	2.3	1.8	0.3
	1205.0^P#	2.8	0.1	111.6	103.4	5.5	5.3^S	1.3

*     Inclusive cells carrying exchanges

^#     Evaluation of 200 metaphases per culture

n.t. Not tested

^P     Precipitation occurred

^S     Aberration frequency statistically significant higher than corresponding control values

^ES   Endomitotic frequency statistically significant higher than corresponding control values

¹     THF    0.5 % (v/v)

²         EMS 900.0 µg/mL

³         CPA     1.4 µg/mL

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, GENOCURE* MBB is considered to be clastogenic in this chromosome aberration test in the presence of S9 mix.

Executive summary:

The test item GENOCURE* MBB, dissolved in tetrahydrofurane (THF), was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamsterin vitro in one experiment. The following study design was performed:

	With and without S9 mix
Exposure period	4 hrs
Recovery	14 hrs
Preparation interval	18 hrs

In each experimental group two parallel cultures were set up. Per culture at least 100 metaphases were scored for structural chromosome aberrations.

The highest applied concentration in the pre-test on toxicity (2410 µg/mL; approx. 10 mM) was chosen with regard to the molecular weight of the test item with respect to the current OECD Guideline 473.

Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation.

No clear dose related toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed up to the highest evaluated test item concentrations.

In the absence of S9 mix no clastogenicity was observed at the concentrations evaluated. In contrast, in the presence of S9 mix two statistically significant and biologically relevant increases in the number of aberrant cells (4.3 and 5.3 %, respectively) were observed after treatment with 150.6 and 1205 µg/mL. These values clearly exceeded the laboratory’s historical control data range (0.0 – 4.0 % aberrant cells, excluding gaps).

No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. In the presence of S9 mix the frequency of endoreduplications was statistically increased after treatment with 301.3 µg/mL.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The substance is non-mutagenic in the in vivo micronucleus assay.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Specific details on test material used for the study:

Identity: GENOCURE* MBB
Chemical Name: Methyl 2-benzoylbenzoate
Batch No.: G07001
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

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Strain: NMRI
Source Harlan Winkelmann GmbH
33178 Borchen,Germany
Number of Animals: 81 (45 males/36 females)
Initial Age at Start of
Acclimatisation: 8 - 10 weeks
Acclimatisation: minimum 5 days
Initial Body Weight
at Start of Treatment:
Mutagenicity experiment:
males mean value 37.5 g (SD +/- 1.6 g)
females mean value 31.2 g (SD +/- 2.1 g)
Bioanalysis:
males mean value 36.7 g (SD +/- 2.2 g)

ENVIRONMENTAL CONDITIONS
Housing: single
Cage Type: Makrolon Type I, with wire mesh top
(EHRET GmbH, D-79302 Emmendingen)
Bedding: granulated soft wood bedding
(Harlan Winkelmann GmbH, D-33178 Borchen)
Feed: pelleted standard diet, ad libitum
(Harlan Winkelmann GmbH, D-33178 Borchen)
Water: tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
Environment: temperature 22  3 °C
relative humidity 30 - 70 %
artificial light 6.00 a.m. - 6.00 p.m.

IN-LIFE DATES: From: April 21, 2008 To: May 07, 2008

Route of administration:

oral: gavage

Vehicle:

Name: 30% DMSO / 70% PEG 400
DMSO Supplier: VWR International (64293 Darmstadt)
Catalogue no.: 1.02931.1000
PEG 400 Supplier: VWR International (64293 Darmstadt)
Catalogue no.: 1.09726.0100
Route and Frequency
of Administration: orally, once
Volume Administered: 10 mL/kg b.w.

Details on exposure:

orally

Frequency of treatment:

once

Remarks:

Doses / Concentrations:
500, 1000, 2000
Basis:

No. of animals per sex per dose:

2 males and 2 females used in the pre-experiment
81 (45 males/36 females) used in the main experiment

Control animals:

yes

Positive control(s):

Name: CPA; Cyclophosphamide
Supplier: Sigma-Aldrich Vertriebs GmbH
82041 Deisenhofen
Catalogue no.: C 0768 (purity: > 98 %)
Dissolved in: deionised water
Dosing: 40 mg/kg b.w.
Route and frequency
of administration: orally, once
Volume administered: 10 mL/kg b.w.

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (Merck, D-64293 Darmstadt)/Giemsa (Merck, D-64293 Darmstadt). Cover slips were mount¬ed with EUKITT (Kindler, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

Evaluation criteria:

Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.
Ten animals (5 males, 5 females) per test group were evaluated as described. The remaining 6th animal of each sex in the respective test group is usually evaluated in case an animal dies in its test group spontaneously.
The study was considered valid as the following criteria are met:
- the negative controls are in the range of our historical control data.
- the positive controls are in the range of our historical control data.
- at least 5 animals per group and sex can be evaluated
- PCE to erythrocyte ratio should not be less than 20 % of the negative control.

Statistics:

(nonparametric Mann-Whitney test)

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

other: see vehicle control

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w. GENOCURE*MBB formulated in 30% DMSO / 70% PEG 400. The volume administered was 10 mL/kg b.w..
The animals treated with 2000 mg/kg b.w. expressed toxic reactions as shown in the table:

toxic hours post-treatment
reactions male / female
1 h 2-4 h 6 h 24 h 30 h 48 h
ruffled fur 2/2 2/0 0/0 0/0 0/0 0/0

On the basis of these data 2000 mg/kg b.w. were estimated to be suitable.

RESULTS OF DEFINITIVE STUDY
In the main experiment for the highest dose group 24 animals (12 males, 12 females) received orally a single dose of 2000 mg/kg b.w. GENOCURE*MBB formulated in 30% DMSO / 70% PEG 400. The volume administered was 10 mL/kg b.w..
The animals treated with 2000 mg/kg b.w. expressed toxic reactions as shown in the table:

Toxic hours post-treatment
hours post-treatment
Reactions male / female
1 h 2-4 h 6 h 24 h 48 h*
ruffled fur 12/12 12/12 12/12 3/1 1/0
*: data only from 6 animals per sex.

For the mid dose group 12 animals (6 males, 6 females) received orally a single dose of 1000 mg/kg b.w. GENOCURE*MBB formulated in 30% DMSO / 70% PEG 400. The volume administered was 10 mL/kg b.w..
The animals treated with 1000 mg/kg b.w. expressed toxic reactions as shown in the table:

toxic hours post-treatment
reactions male / female
1 h 2-4 h 6 h 24 h
ruffled fur 3/4 4/4 4/4 2/1

For the low dose group 12 animals (6 males, 6 females) received orally a single dose of 500 mg/kg b.w. GENOCURE*MBB formulated in 30% DMSO / 70% PEG 400. The volume administered was 10 mL/kg b.w..
The animals treated with 500 mg/kg b.w. expressed toxic reactions as shown in the table:

toxic hours post-treatment
reactions male / female
1 h 2-4 h 6 h 24 h
ruffled fur 0/0 4/3 4/3 2/1

The animals treated with the vehicle control (30% DMSO / 70% PEG 400) did not express any toxic reactions.

 Summary of Micronucleus Test Results

test group		dose mg/kg b.w.		sampling time (h)		PCEs with micronuclei (%)		range		PCE per 2000 erythocytes
vehicle		0		24		0.060		0 -4		1091
test item		500		24		0.075		0 -6		1025
test item		1000		24		0.110		0 -5		1160
test item		2000		24		0.085		0 -3		1176
positive control		40		24		3.205		41 -93		1166
test item		2000		48		0.065		0 -4		1120

1.2   Biometry

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Vehicle control versus test group	Significance	p
500 mg GENOCURE*MBB/kg b.w.; 24 h	-	0.4170
1000 mg GENOCURE*MBB/kg b.w.; 24 h	-	0.0769
2000 mg GENOCURE*MBB/kg b.w.; 24 h	-	0.2041
40 mg CPA/kg b.w.; 24 h	+	< 0.0001
2000 mg GENOCURE*MBB/kg b.w.; 48 h	-	0.5000

      -     =    not significant
      +    =    significant

Conclusions:

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Executive summary:

The test item GENOCURE*MBB was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was formulated in 30% DMSO / 70% PEG 400, which was also used as vehicle control. The volume administered orally was 10 mL/kg b.w.. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

The following dose levels of the test item were investigated:

- 24 h preparation interval: 500, 1000, and 2000 mg/kg b.w..

- 48 h preparation interval: 2000 mg/kg b.w..

As estimated by a pre-experiment 2000 mg GENOCURE*MBB per kg b.w. (the maximum guideline-recommended dose) was suitable. The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that GENOCURE*MBB did not have any cytotoxic properties in the bone marrow. In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with GENOCURE*MBB were near to the value of the vehicle control group. 40 mg/kg b.w. Cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Three in vitro mutagenicity assays and one in vivo mutagenicity assay are available for the substance. The substance is considered to be non-mutagenic based on the results of 3 in vitro assays and one in vivo mutagenicity assay.

Justification for classification or non-classification

The substance was considered to be non-mutagenic based on the results of 3 in vitro mutagenicity assays and one in vivo mutagenicity assay. Therefore, no classification is required according to Regulation EC 1272/2008 and Directive 67/584/EEC.