Methyl 2-benzoylbenzoate

Genetic toxicity: in vivo

Administrative data

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)

Cross-reference

Reason / purpose for cross-reference:

reference to other study

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 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus 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

Constituent 2

Reference substance name:

GENOCURE* MBB

IUPAC Name:

GENOCURE* MBB

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
On the day of the experiment, the test item was formulated in 30% DMSO / 70% PEG 400. The vehicle was chosen to its relative non-toxicity for the animals. All animals received a single standard volume of 10 mL/kg body weight orally.

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

Test animals

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

Administration / exposure

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

Doses / concentrations

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.

Examinations

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)

Results and discussion

Test results

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.

Any other information on results incl. tables

 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

Applicant's summary and conclusion

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.