Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04.07.2014 to 11.12.2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted July 21, 1997
Deviations:
yes
Remarks:
incl. Alkaline Single Cell Electrophoresis Analysis in the Wistar Rat
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
dated May 30, 2008
Deviations:
yes
Remarks:
incl. Alkaline Single Cell Electrophoresis Analysis in the Wistar Rat
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
adopted
September 26, 2014
Deviations:
yes
Qualifier:
according to
Guideline:
other: Minimum Criteria for the acceptance of in vivo alkaline Comet Assay Reports. European Food Safety Authority, EFSA Journal 2012, 10(11): 2977
Principles of method if other than guideline:
Deviations from Study Plan
Deviation No.1:
For the comet assay the Guidelines/Regulations were updated due to a new Guideline.

Deviation No. 2:
For this study Makrolon Type IV was used as cage type instead of II and III as wrongly stated in the study plan.

Deviation No. 3:
The animals of all dose groups were examined for acute toxic symptoms at intervals of around 0-1 h, 2 h and 4 h instead of 1 h and 2-4 h after the third administration of the test item or vehicle.

Deviation No. 4:
The cell suspension of the scraped stomach were not centrifuged as stated in the study plan. These deviations to the study plan, however, do not affect the validity of the study
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
solid
Specific details on test material used for the study:
4-(Methacryloyloxy)benzophenone: 97.14 %
Benzophenone acetate: 1.05 %
4- Hydroxy benzophenone: 1.07 %

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
Test system: Rat (Wistar)
Rationale: Recognised as the recommended test system.
Source: Harlan Laboratories B.V.,Netherlands
Number of animals for the pre-test: -2 males and 2 females
Number of animals for the main study: -42 males
Age: pre-experiment: 8 - 9 weeks (beginning of treatment)
Age: main experiment: 8 - 10 weeks (beginning of treatment)
Body weight:
- first application: 273.5 ± 7.4 g (range: 261.5 – 292.7 g)
- second application: 277.2 ± 8.8 g (range: 263.4 – 301.9 g)
- third application: 286.2 ± 18.4 g (range: 265.7 – 341.3 g)
-
Acclimation:
At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
DMSO / PEG 400 (3/7), was also used as negative control
Details on exposure:
The test item was dissolved in DMSO / PEG 400 (3/7) which was also used as negative control. The animals received the test item, the vehicle (negative control) or positive substances (positive controls) at a constant dose volume of 10 mL/kg b.w. by oral administration.
Duration of treatment / exposure:
The test item was administered three times to seven males per test group at intervals of 48 h, 24 h and 4 h prior to preparation.
Frequency of treatment:
Three times (48, 24 h and 4 h prior to preparation)
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw (total dose)
Dose / conc.:
1 000 mg/kg bw (total dose)
Dose / conc.:
2 000 mg/kg bw (total dose)
No. of animals per sex per dose:
Pre-test: 2 males and 2 females
Main test: 42 males
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- dose: 20 mg/kg

Examinations

Tissues and cell types examined:
Bone marrow erythrocytes (polychromatic erythrocytes)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
dose selection based on acute toxicity pre-test: at 2000 mg/kg bw no toxic signs were found two lower dose groups (500, 1000 and 2000 mg/kg bw) were selected for 24 h treatment interval The administered volume was 10 mL/kg b.w..

DETAILS OF SLIDE PREPARATION:
For the bone marrow isolation tibias/femurs were dissected free of surrounding tissue, the epiphyses was cut off and the marrow flushed out with FBS, using a syringe. The nucleated cells were separated from the erythrocytes using the method of Romagna (Romagna and Staniforth,
1989). Briefly, the cell suspensions were passed through a column consisting of α-Cellulose (Sigma) and Cellulose (Sigmacell type 50). The columns were washed with Hank´s buffered saline. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the
supernatant was discarded. The pellet was re-suspended in a small drop of FBS and spread on slides. The smears were air-dried and then stained with May-Grünwald / Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei by manual inspection. 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, blinded to the evaluator.
The micronucleated PCE’s per 2000 cells and the ratio of polychromatic erythrocytes to the total number of erythrocytes (NCE + PCE) are presented for each animal.

OTHER:
Evaluation criteria:
A test item is classified mutagenic if it induces either a statistically significant dose related increase in the number of micronucleated PCE or a reproducible statistically significant positive result for at least one test point.
Statistics:
Mann-Whitney test

Results and discussion

Test resultsopen allclose all
Sex:
male
Genotoxicity:
negative
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: micronucleus part of this study.
Sex:
male
Genotoxicity:
other: inconclusiv
Remarks:
liver
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Comet in vivo part of the study
Sex:
male
Genotoxicity:
negative
Remarks:
stomach
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Comet in vivo part of the study

Any other information on results incl. tables

Summary of results (Micronucleus part)

 

 

Testgroup

Dose3 times mg/kgb.w.

Sampling time(h)***

PCEswith micronuclei(%)

Range*

PCE per2000 erythrocytes

VehicleControl

0

4

0.257

3 -9

997

Benzophenone methacrylate

500

4

0.193

1 -10

1042

Benzophenone methacrylate

1000

4

0.279

1 -10

1028

Benzophenone methacrylate

2000

4

0.243

1 -7

988

PositiveControl

20**

24

1.207

14 -43

947

* Range of absolute numbers of micronucleated PCEs

** Cyclophosphamide was only applied once

*** Sampling time after the last treatment

 

Summary of results (Comet part):

Analysis of Dead Cell Index and % Tail Intensity in cells of the Liver

 

 

 

TestGroup

Dead cells onslidesper 1500 cells peranimal

Mean ofMedian (% TailIntensity)

GroupMean

SD

GroupMean

SD

VehicleControl

10.52

3.93

3.17

3.08

500 mg/kgb.w. Benzophenone methacrylate

 

10.11

 

4.61

 

5.74

 

3.58

1000 mg/kgb.w. Benzophenone methacrylate

 

9.95

 

3.04

 

5.81

 

2.40

2000 mg/kgb.w. Benzophenone methacrylate

 

10.95

 

4.43

 

8.65

 

4.15

PositiveControl

11.14

6.97

15.80

3.95

 

Summary of results (Comet part):

Analysis of Dead Cell Index and % Tail Intensity in cells of the Stomach

 

 

 

TestGroup

Dead cells onslidesper 1500 cells peranimal

Mean ofMedian (% TailIntensity)

GroupMean

SD

GroupMean

SD

VehicleControl

11.86

4.64

3.90

4.38

500 mg/kgb.w. Benzophenone methacrylate

 

12.24

 

5.72

 

5.99

 

4.59

1000 mg/kgb.w. Benzophenone methacrylate

 

14.90

 

7.89

 

6.78

 

2.95

2000 mg/kgb.w. Benzophenone methacrylate

 

14.00

 

12.24

 

6.12

 

3.03

PositiveControl

18.43

10.65

28.15

7.57

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that under the experimental conditions reported, the test item, up to the maximum dose level tested, did not induce micronuclei as determined by the micronucleus test with bone marrow cells. Therefore, 4-(Methacryloyloxy)benzophenone is considered to be non-genotoxic in the micronucleus part of this study.
Statistically significant increases in primary DNA damage in the liver and stomach as determined by the comet assay were observed, but the values were in the range of the historical vehicle controls. In case of the liver a dose-dependent increase was found. With regard to the results described, the Comet vivo part of this study with the test item 4-(Methacryloyloxy)benzophenone is considered to be inconclusive.
Executive summary:

This study was performed to investigate the potential of 4-(Methacryloyloxy)benzophenone to induce mutagenic / genotoxic effects in the rat by the analysis of micronuclei induction as well as the assessment of single DNA strand breaks in cells isolated from the liver and the stomach. DNA strand breaks were analysed using the alkaline single cell gel electrophoresis (Comet) assay and micronucleus induction was assessed in polychromatic erythrocytes (PCE) using the rat bone marrow micronucleus assay.

The animals received the test item, the vehicle DMSO / PEG 400 (3/7) (negative control) or positive controls at a constant dose volume of 10 mL/kg b.w. by oral administration three times to seven males per test group at intervals of 48 h, 24 h and 4 h prior to

preparation.

After the treatment period liver cells and stomach cells were isolated for the assessment of single DNA strand breaks and bone marrow cells were collected for micronuclei analysis at concentrations of 500, 1000 and 2000 mg/kg b.w..

 

Micronucleus assay – Bone Marrow

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. After treatment with the test item the number of PCEs in the bone marrow was not substantially decreased as compared to the mean value of PCEs of the negative control thus indicating that 4-(Methacryloyloxy)benzophenone did not exert any cytotoxic effects in the bone marrow.

At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. In

comparison to the corresponding vehicle control there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei after administration of the test item and with any dose level used.

20 mg/kg b.w. cyclophosphamide administered once orally was used as positive control which showed a substantial and statistically significant increase of induced micronucleus frequency demonstrating the validity of this part of the study.

 

Comet assay – Liver

As a cytotoxic parameter the dead cell index (dead cells (apoptotic/necrotic cells) per 1500 nuclei per animal) was determined in the Comet assay and did not show any increased cytotoxic effects in the liver cells in comparison to the vehicle control.

For the analysis of the single DNA strand breaks 150 cells per animal (3 slides prepared for each animal, 50 cells evaluated per slide) were evaluated. The relevant parameter for DNA damage is the percentage of DNA in the Comet tail measured as intensity relative to the intensity of the nucleus (% Tail intensity).

 

The Comet assay on cells of the liver revealed distinct and statistically significant increases in

DNA damage at any of the tested dose levels compared to the corresponding vehicle controls on the evaluated parameter (% Tail intensity). Additionally, a dose-dependent increase could be observed. However, the values were still in the range of the historical vehicle control data.

 

Comet assay – Stomach

As a parameter for cytotoxicity the dead cell index (dead cells (apoptotic/necrotic cells) per 1500 nuclei per animal) was determined in the Comet assay and did not show any increased cytotoxic effects in the stomach cells in comparison to the vehicle control.

For the analysis of the single DNA strand breaks 150 cells per animal (3 slides prepared for each animal, 50 cells evaluated per slide) were evaluated. The relevant parameter for DNA damage is the percentage of DNA in the Comet tail measured as intensity relative to the intensity of the nucleus (% Tail intensity).

The Comet assay on cells of the stomach revealed distinct and statistically significant increases in DNA damage at any of the tested dose levels compared to the corresponding vehicle controls on the evaluated parameter (% Tail intensity). However, all obtained test item group values were clearly in the range of the historical vehicle control group and no dose-dependency could be observed.

For both tissues, the vehicle controls were in the range to ensure a valid performance of the study.

The reference mutagen [MMS, 25 mg/kg b.w. oral] showed a distinct and statistically significant increase of DNA damage as detected by % Tail intensity analysis.

 

In conclusion, it can be stated that under the experimental conditions reported, 4-(Methacryloyloxy)benzophenone, up to the maximum dose level tested, did not induce micronuclei as determined by the micronucleus test with bone marrow cells. Therefore, 4-(Methacryloyloxy)benzophenone is considered to be nongenotoxic in the micronucleus part of this study.

Statistically significant increases in primary DNA damage in the liver and stomach as determined by the comet assay were observed, but the values were in the range of the historical vehicle controls. In case of the liver a dose-dependent increase was found. With regard to the results described, the Comet vivo part of this study with the test item 4-(Methacryloyloxy)benzophenone is considered to be inconclusive.