2-methylenepropane-1,3-diyl diacetate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Vivo Comet Test including Micronucleus Analysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 06 February 2014 and 13 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was assigned Reliability 1 as it was conducted to OECD guidelines and in compliance with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

other: Burlinson, B., Tice, R.R., Speit, G., Agurell, E., Brendler-Schwaab, B., Collins, A., Escobar, P., Honmah, M., Kumaravel, T., Nakajima, M., Sasaki, Y.F., Thybaud, V., Unom, Y., Vasquez, M., Hartmann, A. (2007). Fourth International Workgroup on Genotoxici

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

other: Tice, R.R., Agurell E., Anderson D., Burlinson B., Hartman A., Kobayashi H., Miyame Y., Rojas, E. Ryu J.-C. and Sasaki Y.F. (2000). Single Cell Gel/Comet Assay: Guidelines for in vitro and in vivo genetic toxicology testing. Environmental & Molecular Muta

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

other: Hartmann, A., Augurell, E., Beevers, C., Brendler-Schwaab, S., Burlinson, B., Clay P., Collins, A., Smith, A., Speit, G., Thybaud, V. and Tice, R.R.; 2003; Recommendations for conducting the in vivo alkaline Comet assay. Mutagenesis, vol 18, no.1, pp. 45-

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

EC No. 440/2008

Deviations:

no

Test guideline 6

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

Test guideline 7

Qualifier:

according to guideline

Guideline:

other: US FDA (Red book 2000), Toxicological Principles fur the Safety Assessment of Food Ingredients. IV.C. I .d. Mammalian Erythrocyte Micronucleus Test.

Deviations:

no

Test guideline 8

Qualifier:

according to guideline

Guideline:

other: ICH (2011) EMA/CHMI't1CH/126642/2008. Guideline S2(Rl): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

2-methylenepropane-1,3-diyl diacetate

EC Number:

223-225-8

EC Name:

2-methylenepropane-1,3-diyl diacetate

Cas Number:

3775-29-9

Molecular formula:

C8H12O4

IUPAC Name:

2-[(acetyloxy)methyl]prop-2-en-1-yl acetate

Specific details on test material used for the study:

Identity: 2-Methylene-l ,3-propanediol diacetate
Molecular weight: 172.2
Lot number: 201210
Expiry: December 2017
Appearance: Colorless liquid
Storage conditions: Room temperature, in the dark
Purity/ Assay: >99%
Correction factor for formulation: None, to be used as supplied
Intended commercial use: Food contact materials

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

All animals used on this study were CD-1 mice.
On the day after arrival from Charles River UK Limited, Margate, Kent, England, animals used on the study weighed between:
Preliminary toxicity test: Males weighed between 28.6 g and 31 .8 g
Females weighed between 23.3 g and 26.0 g
Micronucleus test· Males weighed between 27.9 g and 35.2 g.

Animal age on dispatch and on Day 1 of dosing was:
Preliminary toxicity test. On Dispatch: Males and females ca 35-42 days old.
Day 1: Males and females ca 40-54 days old.
micronucleus test: On Dispatch: Males and females ca 40-54 days old.
Day l: Males ca 40-47 days old

After arrival the weight of the animals was checked and found to be acceptable. The animals were randomly assigned to groups and given a unique tail tattoo. The animals were maintained in a controlled environment with the thermostat and relative humidity target ranges set at 19 to 23'C and 40 to 70% respectively. The room was illuminated by ai1ificial light for 12 hours per day.
All animals were allowed free access to pelleted eXpanded rat and mouse No. I maintenance diet (SQC grade obtained from Special Diets Services Ltd, Witham, EssBX, UK) and tap water ad libitum.
Food and tap water are routinely analysed for quality at source. All animals were given small soft white untreated wood (ASPEN) chew blocks and a red plastic shelter fur environmental en1ichment, and they were acclimatized fur a minimum of 5 days.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Corn oil

Details on exposure:

Test substance formulation and administration
Suspensions of the test substance were prepared in corn oil, supplied by Sigma, batch numbers MKBL8756V and MKBP7039V.
Mitomycin C obtained from Sigma, batch number SLBH9654V was used as the positive control compound for the micronucleus phase of the study. A solution was prepared using purified water at a concentration of 0.6 mg/mL
Ethyl methanesulphonate (EMS) obtained from Sigma, batch number BCBM2272V was used as the positive control compound for the Comet phase of this study. A solution was prepared using purified water at a concentration of 20 mg/mL just prior to administration.
All animals in the vehicle control and test substance dose groups were dosed orally by gavage using a dose volume of 10 mL/kg. The vehicle control group received corn oil. The positive control group for the micronucleus phase of the study were administered Mitomycin C at 12 mg/kg using a dose volume of 20 mL/kg. The positive control group for the Comet phase of the study were administered Ethyl methane sulphanate at 200 mg/kg using a dose volume of 10 mL/kg.
Study Design
Following the preliminary toxicity test no substantial differences in toxicity were observed between sexes. Therefore in line with current guidelines, the comet test was performed using male mice only.
From the results obtained in the preliminary toxicity study, dose levels of 62.5, 125 and 250 mg/kg/day were used for the comet test.

Following dosing, the animals were examined regularly during the working day for a period of 24 hours after the first dose, 21 hours after the second dose and after the third dose any mortalities or clinical signs of reaction during the experiment were recorded.
Animals from the vehicle control and test substance groups were culled 3 hours after administration of the third dose. In addition, animals in the positive control groups were culled 3 hours (comet positive control) and 24 hours (micro nucleus positive control) after a single dose. Animals were culled by exposure to rising levels of carbon dioxide.

Duration of treatment / exposure:

Animals were treated with MPDA orally by gavage on three occasions, the second dose being administered approximately 24 hours after the first dose, with the third dose being administered approximately 21 hours after the 2nd dose, 3 hours before sampling

Frequency of treatment:

See above

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

62.5 other: mg/kg/day

Doses / concentrations 2

Dose / conc.:

125 other: mg/kg/day

Doses / concentrations 3

Dose / conc.:

250 other: mg/kg/day

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethyl methanesulphonate (EMS) obtained from Sigma, batch number BCBM2272V was used as the positive control compound for the Comet phase of this study. A solution was prepared using purified water at a concentration of 20 mg/mL just prior to administration

Examinations

Tissues and cell types examined:

Comet Phase: Duodenum and liver
Micronucleus Phase: Bone marrow from femurs

Details of tissue and slide preparation:

Comet Phase
Sections of the duodenum and liver were placed into ice cold mincing solution, all samples were stored on ice before processing for Comet analysis. Single cell suspensions were prepared using a tissue specific method.
Comet slides were prepared from all cell suspensions.
Sections of the duodenum and liver were stored in 10% buffered formalin. On finalisation of the study report, these tissues (if not required) will be disposed of and the slides archived.

Micronucleus Phase
Both femurs were dissected out from each animal. The femurs were cleaned of all excess tissue and blood and the proximal epiphysis removed from each bone. The bone marrow of both femurs from each animal was flushed out and pooled in a total volume of 3 mL of filtered foetal calf serum by aspiration.
The resulting cell suspensions were centrifuged at 1000 rpm (150 x g) for 5 minutes and the supernatant discarded. The final cell pellet was resuspended in a small volume of foetal calf serum to facilitate smearing in the conventional manner on glass microscope slides (Schmid 1976).

Slide preparation/Fixation and slide staining
Comet Phase
Glass slides were dipped in 1 % normal melting point agarose and left to air dry prior to addition of the cell suspension layer.
For each tissue type, an appropriate dilution of the cell suspensions were made and mixed with the appropriate volume of 0.5% low melting point agarose. A 75 µL aliquot of the cell/agar mix was dispensed onto the appropriate pre-dipped slides and cover-slipped.
Once the agar had set the cover slips were removed and the slides immersed in chilled lysis solution in a light proof box. These were stored refrigerated overnight prior to electrophoresis

Micronucleus Phase
1 Fixed for a minimum of 10 minutes in methanol and allowed to air-dry
2 Rinsed in purified water
3 Stained in acridine orange solution (0.0125 mg/mL using purified water) for 4 minutes
4 Washed in purified water for 5 minutes
5 Rinsed in with tap water for 2 minutes
6 Stored at room temperature until required
7 Immediately prior to scoring, slides are wet mounted with coverslips using purified water

Electrophoresis
Comet Phase
The slides were placed onto a dry, level platform of a horizontal electrophoresis unit containing chilled electrophoresis buffer. The slides from each tissue from each treatment group were spread across the platform between separate electrophoresis units to avoid any positional electrophoretic effects.
The buffer reservoir was filled with electrophoresis buffer until the slides were covered and the nucleoids were left to unwind for 20 minutes at approximately 2-8"C.
After alkali unwinding the slides were electrophoresed at 18V, approximately300mA (between 0.7 and 1.0 V/cm) for 30 minutes.
Once electrophoresis was complete the slides were washed with neutralising buffer (3 x 5 minutes) and stored, refrigerated in lightproof boxes with moistened tissue paper to prevent agar dehydration.

Microscopic examination
Comet Phase
Slides were coded as per protocol. Slides were examined by staining with SYBR GOLD® and visualised under a fluorescence microscope. The comet images from the microscope were projected onto a computer display screen via a CCD camera and measured using Perceptive Instruments COMET IV™ image analysis system.

Initially the slides were examined for any overt toxicity, e.g. an increase in background debris and/or an increase in the incidence of excessively damaged cells (i.e. 'hedgehog' or 'ghost' comets). These cells were excluded from the analysis, along with ·any cells that had unusual staining artefacts.
Fifty cells were scored per slide to give a total number of 150 cells per tissue per animal. The extent of DNA migration and hence damage is reflected by:

% TAIL INTENSITY, defined as the fluorescence detected by image analysis in the tail, which is proportional to the amount of DNA that has moved from the head region into the comet tail.

Micronucleus Phase
Coded slides were examined by fluorescence microscopy and 2000 polychromatic erythrocytes per animal were examined for the presence of micronuclei. One smear was examined per animal, any remaining smears being held temporarily in reserve in case of technical problems with the first smear.
The proportion of polychromatic erythrocytes was assessed by examination of a total of at least 1000 erythrocytes per animal and the number of micronucleated normochromatic erythrocytes was recorded.

Evaluation criteria:

Acceptance criteria
Comet Phase
The following criteria were applied fur assessment of assay acceptability:
l. The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database
2. Concurrent positive control should induce responses that are compatible with those generated in the historical positive control database and produce a statistically significant increase compared with the concurrent negative control

Micronucleus Phase
The following criteria were applied for assessment of assay acceptability:
1. Each treated and control group should include at least 5 analysable animals.
2. Vehicle control values for micronucleated polychromatic erythrocytes must be consistent with the laboratory historical vehicle control data.
3. Positive controls must show clear unequivocal positive responses.
4. When analysing slides the proportion of polychromatic erythrocytes should not be less than 20% of the vehicle control value.

Statistics:

See "Any othe information" below

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Preliminary toxicity test

Clinical signs

To determine suitable dose levels for use in the main test, three groups consisting of two male and two female animals were administered MPDA at either 1000, 500 or 250 mg/kg/day on three occasions, the second dose being administered approximately 24 hours after the first dose, with the third dose being administered approximately 21 hours after the second dose, 3 hours before termination.

At 1000 mg/kg/day clinical signs of toxicity observed included hunched posture, piloerection, underactive behaviour reduced body temperature, reduced body tone and both eyelids partially closed. One male animal and both female animals were found dead after dosing on Day 1, the remaining male animal was killed in extremis due to the severity of the clinical signs observed. The maximum tolerated dose was therefore exceeded at 1000 mg/kg/day.

At 500 mg/kg/day clinical signs of toxicity observed included piloerection, hunched posture, underactive behaviour, hindlimbs splayed, prostrate posture, slow breathing, shallow breathing, eyelids partially closed, reduced body temperature and reduced body tone. One male animal and one female animals were killed in extremis due to the severity of the clinical signs observed the remaining male and female animals were killed as the dose level was considered unacceptable for the main test. The maximum tolerated dose was therefore exceeded at 500 mglkgfday.

At 250 mg/kg/day clinical signs of toxicity observed included piloerection, hunched posture, underactive behaviour, both eyelids partially closed and pallor skin colour. All animals survived until scheduled termination on day 3.

Some incidences of bodyweight loss weer observed throughout the preliminaiy toxicity testing.

The post mortem examinations did not find any signs of mis-dosing.

MPDA administered at 250 mglkgfday was therefore considered to be the maximum tolerated dose in both male and female animals. No substantial differences in toxicity were observed between the sexes, therefore, in line with current guidelines the main test was performed using male animals only, dose levels of 62.5, 125 and 250 mg/kg/day were selected.

 

Main test

The main test was carried out in male animals only

Clinical signs

Animals were treated with MPDA at dose levels of 62.5, 125 and 250 mg/kg/day. No clinical signs of toxicity were observed over the duration of the test. However one male animal was found dead on Day 2 after receiving one dose of MPDA at 250 mg/kg/day.

Some incidences of bodyweight loss were observed throughout the main testing.

The post mortem examination did not find any signs of mis-dosing.

 

% Tail intensity

No statistically significant increases in the median % TI were observed in the liver of male CDf mice administered MPDA at any dose level, compared to vehicle control values.

No statistically significant increases in the median % TI were obsered in the duodenum of male CDl mice administered MPDA at 62.5 mg/kg(day, compared to vehicle control values

Statistically significant increases in the median% TI were observed in the duodenum of male CDI mice administered MPDA at 125 and 250 mg/kg/day, compared to vehicle control values (p<0.05, Williams' test). However this result is not considered to be biologolically relevant as mean% Tl values fall within or close to current vehicle historical control ranges.

The positive control compound, Ethyl methanesulphonate, produced statistically significant increase, in the median % TI when compared to vehicle control values (p<0.001, t-test).

 

Hedgehog "Ghost" cell data

No Hedgehog "Ghost" cells were observed in either the duodenum or liver of male CD-1 mice.

 

Micronucleated polychromatlc erythrocyte counts (MPCE)

No statistically significant increases in the frequency of micronucleated polychromatic erythrocytes were observed in male CD-1 mice administered MPDA at 125 or 250 mg/kg/day when compared to control values. However a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes was observed in male CD-1 mice administered MPDA at 62.5 mg/kg/day (p<0.05, pairwise). This result is not considered to be biologically relevant as micronucleated polychromatic erythrocyte values fall within the current vehicle historical control ranges.

The positive control compound, Mitomycin C produced a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes (p<0.01, pairwise)

 

Micronucleated norrnochromatlc erythrocytes (MNCE)

MPDA did not cause any significant increases in the incidence of micronucleated normochromatic erythrocytes in male CDI mice.

 

Proportion of polychromatic erythrocytes (%PCE)

MPDA did not cause any statistically significant decrea"ses in the proportion of polychromatic erythrocytes in male CDI inice.

Applicant's summary and conclusion

Conclusions:

Comet Phase
It is concluded that MPDA has not shown any evidence of causing an increase in DNA strand breaks or cytotoxicity in the duodenum or liver of male CD-1 mice when administrated orally by gavage in this in vivo test procedure.
Micronucleus Phase
It is concluded that MPDA has not shown any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male CD-1 mice when administrated orally by gavage in this in vivo test procedure.

Executive summary:

This study was designed to assess the potential of 2-Methylene-1,3-propanediol diacetate (MPDA) to induce DNA strand breaks in the duodenum or liver of CD1 mice and also assess the potential induction of micronuclei by MPDA in the bone marrow cells of CD1 mice.

Animals were treated with MPDA orally by gavage on three occasions, the second dose being administered approximately 24 hours after the first dose, with the third dose being administered approximately 21 hours after the secnd dose, 3 hours before sampling.

On the basis of results from the preliminary toxicity test, dose levels of 62.5, 125 and 250 mg/kg/day were selected for the assay. No substantial differences in toxicity were observed between the sexes in the preliminary toxicity test, therefore, in line with current guidelines the comet test was performed using male animals only.

All animals in the vehicle control and test substance dose groups were dosed orally by gavage using a dose volume of 10 mL/kg. The vehicle control group received corn oil. The positive control group for the micronucleus phase of the study were administered Mitomycin Cat 12 mg/kg using a Close volume of 20 mL/kg. The positive control group for the comet phase of the study were administered Ethyl methanesulphonate at 200 mg/kg using a dose volume of 10 mL/kg.

 

Comet Phase

Cell suspensions from each tissue were obtamed from male animals in the vehicle control group and in each of the test substance groups 3 hours after administration of the third dose. Cell suspensions from male animals in the positive control group were obtained approximately 3 hours after a single dose.

Following electrophoresis three slides per animal per tissue were analysed for comets. Slides were visualised by staining with SYBR GOLD® via fluorescence microscopy and 150 morphologically normal cells were analysed for the presence of comets per tissue per animaL

DNA strand breaks were assessed by comparing the mean and median % tail intensities (% TI) from MPDA treated animals compared with vehicle control values. The slides were also examined for any overt toxicity, e.g. an increase ln background debris and/or an increase in the incidence of excessively damaged cells (i.e. 'hedgehog' or 'ghost' cells). These cells were excluded from the analysis, along with any cells that had unusual staining artefacts.

 

Micronucleus Phase

Bone marrow smears were obtained from animals in the vehicle control and in each of the test substance groups 3 hours after administration of the third dose. In addition, bone marrow smears were also obtained from animals in the positive conrrol group 24 hours after a single dose.

One smear from each animal was examined for the presence of micronuclei in 2000 polychromatic erythrocytes. The proportion of polychromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated nonnochromatic erythrocytes was also kept.

 

Results

Comet Phase

No statistically significant increases in the median % TI were observed in the liver of male CD1 mice administered MPDA at any dose level, compared to vehicle control values.

No statistically significant increases in the median % TI were observed in the duodenum of male CDl mice administered MPDA at 62.5 mg/kg/day, compared to vehicle control values.

Statistically significant increases in the median % TI were observed in the duodenum of male CD1 mice administered MPDA at 125 and 250 mglkg/day, compared to vehicle control values (p<0.05, Williams' test). However this result is not considered to be biologically relevant as mean % TI values fall within or close to current vehicle historical control ranges.

The positive control compound, Ethyl methanesulphonate, produced statistically significant increases in the median % TI when compared to vehicle control values (p<0.001, !-test).

No Hedgehog "Ghost' cells were observed in either the duodenum or liver of male CD1 mice.

Micronucleus Phase

No statistically significant increases in the frequency of micronucleated polychromatic erythrocytes were observed in male CD1 mice administered MPDA at 125 or 250 mg/kg/day when compared to control values. However a statistically significant increase in the frequency of micronucleatecl polychromatic erythrocytes were observed in male CD1 mice administered MPDA at 62.5 mg/kg/day (p<0.05, pairwise) This result is not considered to be biologically relevant as micronucleated polychromatic erythrocyte values fall within the current vehicle historical control ranges.

No statistically significant decreases in the proportion of Polychromatic erythrocytes were observed in male CD1 mnice administered MPDA at any dose level, compared to vehicle control values.

The positive control compound, Mitomycin C produced a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes (p<0.01, pairwise).

 

Conclusions

Comet Phase

It is concluded that MPDA has not shown any evidence of causing an increase in DNA strand breaks or cytotoxicity in the duodenum or liver of male CD1 mice when administrated orally by gavage in this in vivo test procedure.

Mlcronucleus Phase

It is concluded that MPDA has not shown any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male CD1 mice when administrated orally by gavage in this in vivo test procedure.