2-methylenepropane-1,3-diyl diacetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial Reverse Mutation Test In vitro (Huntingdon Life Sciences, 2013, LYA0037): it was concluded that the test item showed no evidence of mutagenic activity under the conditions of this test and according to OECD Test Guideline 471.

In vitro Mammalian Chromosome Aberration Test In Human Lymphocytes, (Huntingdon Life Sciences, 2013, LYA0041): conducted in compliance with OECD Guideline 473, it was concluded that 2-Methylene-1,3-propanediol diacetate has shown evidence of causing an increase in the frequency of stmctural chromosome aberrations, in the absence of S9 mix only following 21-hout continuous treatment, under the experimental conditions described.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 19 February 2013 and 18March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability 1 is assigned because the study is conducted according to OECD TG 471, in compliance with GLP, without deviations that influence the quality of the results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

EC No. 440/2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US Food and Drug Administration. Toxicological Principles for the Safety Assessment of Food Ingredients (Redbook 2000): IV.C.1.a. Bacterial Reverse Mutation Test.

Deviations:

no

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Official notice ofMHLW, ME TT and MOE (31 March 2011) YAKUSHOKUHATSU0331 No7 SETKYOKU No 5 KANPOKTHATSUNo 110331009

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: 2-Methylene-1,3-propanediol diacetate
Lot number: 1210
Expiry: December 2017
Appearance: Colour less liquid
Storage conditions: Room temperature, in the dark
Purity/ Assay: >99%
Date received: 31 January 2013

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

- First Test:
The following dose levels were used (all strains): 5, 15, 50, 150, 500, 1500, 5000 µg/plate

- Experiment 2:
All strains: 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

The Sponsot indicated that 2-Methylene-1,3-propanediol diacetate was insoluble in water but that dimethyl sulphoxide (DMSO) was a suitable solvent DMSO (HPLC grade) was, therefore, used as the vehicle for this study.
The highest concentration of2-Methylene-l,3-ptopanediol diacetate tested in this study was 50 mg/mL in the chosen vehicle, which provided a final concentrntion of 5000 µg/plate .. This is the standard limit concentration recommended in the regulatmy guidelines that this assay follows .. The highest concentration in each test was diluted with DMSO to produce a series of lower concentrations, separated by apptoximately half-log10 intervals.
All concentrations cited in this repott ate expressed in terms of the 2-Methylene-1,3propanediol diacetate sample as received.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

100 µL/plate DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see section "Any other information on materials and methods incl. tables"

Details on test system and experimental conditions:

S9 metabolizing system
Preparation of S9 fraction
S9 fraction, prepared from male Sprague-Dawley derived rats, dosed i.p. with phenobarbital sodium (30 mg/kg 4 days before killing and 60 mg/kg I, 2 & 3 days before killing) and 5,6benzoflavone (80 mg/kg 2 days before killing) to stimulate mixed-function oxidases in the liver, was purchased from a commercial source and stored at approximately -80°C. The quality control statement relating to each batch of S9 preparation used is included in the raw data
Lot No .. : 2946 (Date of preparation: 25 May 2012)

Preparation of S9 mix
The S9 mix contained: S9 fraction (10% v/v), MgCh (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADPH (4 mM) and NADH (4 mM) in water.. All the cofactors were filter-sterilised befote use

Mutation test procedure
First test
Aliquots ofO I mL ofthe test substance solutions (seven concentrations up to 5000 µg/plate), positive control 01 vehicle control were placed in glass tubes. The vehicle control was DMSO. S9 mix (0.5 mL) or 0 1 M pH 7 4 phosphate buffer (0.5 mL) was added, followed by 0.1 mL ofa 10-hour bacterial culture and 2 ml of agar containing histidine (0 05 mM), biotin (0 .. 05 mM) and tryptophan (0 .. 05 mM).. The mixture was thoroughly shaken and overlaid onto previously prepared Petli dishes containing 25 mL minimal agar. Each Petri dish was individually labelled with a unique code, identifying the contents of the dish Three Petri dishes were used for each treatment Plates were also prepated without the addition of bacteria in order to assess the sterility ofthe test substance, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37°C for ca 72 hours .. After this period, the appearance of the background bacterial lawn was exainined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer)
Any toxic effects of the test substance may be detected by a substantial reduction in mean revertant colony counts, by a sparse or absent background bacterial lawn, or both. In the absence of any toxic effects, the maximum concentration selected for use in the second test is the same as that used in the first If toxic effects are observed, a lower concentration might be chosen, ensuring that signs of bacterial inhibition are present at this maximum concentration .. Ideally, a minimum of four non-toxic concentrations should be obtained .. If precipitate is observed on the plates at the end of the incubation period, at least one precipitating concentration should be included in the second test, unless otherwise justified by the Study Director

Second test
As a cleat negative response was obtained in the fast test, a vaiiation to the test procedure was used for the second test The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37°C for 30 minutes with shaking before the addition ofthe agar overlay. The maximum concentration chosen was again 5000 µg/plate, but only five concentrations were used.
Stability, homogeneity and formulation analysis
The stability of 2-Methylene-l,3-propanediol diacetate and the stability and homogeneity of 2-Methylene-1,3-prnpanediol diacetate in the vehicle were not determined as part of this study. Analysis of achieved concentration was not performed as part of this study.

Analysis of data
The mean number and standard deviation of revertant colonies were calculated for all groups. The “fold-increases” relative to the vehicle controls were calculated in order to compare the means for all treatment groups with those obtained for the vehicle control groups.

Criteria for assessing mutagenic potential
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concunent vehicle controls, with some evidence of a positive concentration-response telationship, it is considered to exhibit mutagenic activity in this test system.
If exposure to a test substance does not prnduce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system No statistical analysis is pet fmmed.
If the 1esults obtained fail to satisfy the criteria for a clear "positive" OJ "negative" response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers .. The statistical prncedures used are those described by Mahon et al (1989) and are usually Dunnett's test followed, if apprnpriate, by trnnd analysis .. Biological impmtance will be considered along with statistical significance. In general, treatment-associated increases in revertant colony numbers below two or three times those of the vehicle cont10ls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained

Evaluation criteria:

Acceptance criteria
For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the current historical control range for the laboratory unless otherwise justified by the Study Director. The historical range is maintained as a rolling record over a maximum of five years. Also, the positive control compounds must induce an increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537, which have relatively low spontaneous reversion rates) that of the concurrent vehicle controls. Mean viable cell counts in the 10-hour bacterial cultures must be at least 10^9/mL.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The absence of colonies on sterility check plates confirmed the absence of microbial contamination of the S9 mix, buffer and test substance formulation
The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory.. Appropriate positive control chemicals (with S9 mix where requiied) induced substantial increases in revertant colony numbers with all strains in all repmted tests, confirming sensitivity of the cultures and activity of the S9 mix

First test
No evidence of toxicity was obtained following exposure to 2-Methylene-1,3-propanediol diacetate. A maximum exposure concentration of 5000 µg/plate was, therefore, selected for use in the second test.
No substantial increases in revertant colony numbers over control connts were obtained with any of the tester strains following exposure to 2-Methylene-1,3-propanediol diacetate at any concentration up to and including 5000 µg/plate in either the presence or absence of S9 mix.

Second test
No evidence of toxicity was obtained following exposure to 2-Methylene-l, 3-propanediol diacetate.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to 2-Methylene-1,3-propanediol diacetate at any concentration up to and including 5000 µg/plate in either the presence 01 absence of S9 mix

Remarks on result:

other:

Remarks:

no evidence of mutagenic activity

 

 

Conclusions:

It was concluded that 2-Methylene-1,3-propanediol diacetate showed no evidence of mutagenic activity in this bacterial system under the test conditions employed

Executive summary:

In this in vitro assessment of the mutagenic potential of 2-Methylene-l,3-propanediol diacetate, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains IA1535, IA1537, IA98andIA100, and a t1yptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKMIOI), were exposed to 2-Methylene-1,3-propanediol diacetate diluted in dimethyl sulphoxide (DMSO). DMSO was also used as a vehicle control.

Two independent mutation tests were performed in the presence and absence oflive1 preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first test was a standard plate incorporation assay; the second included a pre-incubation stage.

Concentrations of2-Methylene-l,3-propanediol diacetate up to 5000 µg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration.

No signs of toxicity towards the teste1 strains were observed in either mutation test following exposure to 2-Methylene-1,3-propanediol diacetate.

No evidence ofmutagenic activity was seen at any concentration of2-Methylene-l,3propanediol diacetate in either mutation test

The concurrent positive controls ve1ified the sensitivity of the assay and the metabolising activity of the liver preparations The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratmy

It was concluded that 2-Methylene-1,3-propanediol diacetate showed no evidence of mutagenic activity in this bacterial system under the test conditions employed

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 16 July 2013 and 04 September 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well conducted and well described study in accordance with GLP and OECD guideline 473 without any deviation

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EC No. 440/2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US FDA

Version / remarks:

US Food and Drng Administration. Toxicological Principles for the Safety Assessment ofFood Ingredients (Redbook 2000): IV.C.Lb. In vitro Mammalian Chromosome Aberration Test.

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Official notice ofMHLW, MEII and MOE (31 March 2011) YAKUSHOKUHATSU 0331No7 SEIKYOKU No 5 KANPOKTHATSUNo 110331009.

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Identity: 2-Methylene-1,3-propanediol diacetate (MPDA)
Moleculat weight: 1722
Batch number: 201210
Expiry: December 2017
Appearance: Colorless liquid
Storage conditions: Room temperature in the dark
Purity/Assay:>99%
Date received: 31Januaty2013

Species / strain / cell type:

lymphocytes: cultures prepared from the pooled blood of two healthy, non-smoking, adult donors

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction, prepared from male Sprague-Dawley derived rats, dosed i.p. with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was purchased from a commercial source and stored at ca -80°C.

Test concentrations with justification for top dose:

The final concentrations to which cells were exposed initially are given below. Those cultures that were analysed for chromosome aberrations areindicated *.
Preliminary toxicity test: 17.35, 258.92, 48.20, 80.34, 133.90, 233.17, 371.95, 619.92, 1033.20 and 1722 µg/mL
Main tests: -S9 mix (3 hours) 233.17, 371.95, 619.92, 1033.20 and 1722 µg/mL
+S9 mix (3 hours) 233.17, 371.95, 619.92, 1033.20 and 1722 µg/mL
-S9 mix (21 hours) 233.17, 371.95, 619.92, 1033.20 and 1722 µg/mL

Vehicle / solvent:

Selection of vehicle and formulation of test substance
2-Methylene-l, 3-propanediol diacetate was miscible in DMSO at 172 .2 mg/ml. On dosing a 172.2 mg/mL solution at 1% v/v into aqueous tissue culture medium, giving a final concentration of 1722 µg/mL (10 mM), no notable culture medium changes were observed, when compared to the vehicle control
Concentrations with high ionic strength and osmolality may cause chromosomal aberrations (Galloway et al. 1987}. Therefore, the osmolality and pH ofthe test substance in medium was tested at 1722 µg/mL. No fluctuation in osmolality of more than 50 mOsm/kg and no change in pH of more than I.0 unit were observed, when compared to the vehicle control.
In this case, the highest final concentration used for subsequent testing was 1722 µg/mL (10 mM), as this is the standard limit concentration within this test system as 1ecommended in the regulatory guidelines ..
In this study 2-Methylene-1,3-propanediol diacetate was added to cultures at 1% v/v (50 µL pe1 5 mL culture).
All concentrations cited in this report are exp1essed in terms of 2-Methylene-1,3-propanediol diacetate sample as received.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

Preliminary toxicity test procedure
Cultures were treated approximately 48 hours after commencement of incubation of lymphocyte cultures. All cultures were uniquely identified. Cultures were prepared for each treatment (3-hour treatment in the absence and presence of S9 mix, and 21-hour continuous treatment in the absence of S9 mix).
Duplicate cultures were used for treatment with the vehicle, and single cultures for treatment with the test substance for each test condition. No positive control cultures were prepared.
All cultures were centrifuged and resuspended in fresh medium before treatment, and S9 homogenate was present in appropriate cultures at a final concentration of 5% v/v.
2-Methylene-1,3-propanediol diacetate was added to each culture in 50 µL aliquots to give final concentrations of 17.35, 28..92, 48.20, 8034, 133 90, 233 17, 371.95, 619 . .92, 1033.20 and 1722 µg/mL DMSO was used as the vehicle control.
At the end of the 3-hour treatment period, cultures were examined for the presence of precipitate. Cultures were then centrifuged at 500g for 5 minutes and the supernatant removed. Cultures were then washed in saline (5 mL), resuspended in fresh medium, and incubated (for approximately 18 hours) until the scheduled harvest time. The cultures were then harvested, slides prepared, and microscopically examined for cytotoxicity using a light microscope.
At the end of the 21-hour treatment period, cultures were examined for the presence of precipitate and any observations noted in the study file The cultures were then hmvested, slides prepared, and microscopically examined for cytotoxicity using a light microscope

Harvesting and fixation
Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid® to each culture at a final concentration of 0.1 µg/mL. After 2 hours incubation, each cell suspension was transferred to a centrifuge tube and centrifuged for 5 minutes at 500g. The cell pellets were treated with a hypotonic solution (0.075M KCl), pre-warmed at 37°C. After a 10 minute period of incubation at 37°C, the suspensions were centrifuged at 500g for 5 minutes and the cell pellets fixed by addition of freshly prepared cold fixative (3 parts methanol : 1 part glacial acetic acid). The fixative was replaced until it was clear.

Slide preparation
The pellets were resuspended, then centrifuged at 500g for 5 minutes and finally resuspended in a small volume of fresh fixative. A few drops of the cell suspensions were dropped onto pre-cleaned microscope slides and allowed to air dry. The slides were then stained in 10% Giemsa, prepared in buffered water (pH 6.8). After rinsing in buffered water the slides were left to air-dry and mounted in DPX. The remainder of the cell pellets in fixative were stored at approximately 4°C until slide analysis was completed.

Microscopic examination for cytotoxicity
The prepared slides were examined by light microscopy and the incidence of mitotic cells per 1000 cells assessed. Slides were assessed for a depression in mitotic index (except when clear evidence of overt toxicity is observed, or in cultures where there were no signs of cytotoxicity).
In the absence ofS9 mix following 3-hour treatment, 2-Methylene-1,3-propanediol diacetate caused no reduction in the mitotic index at 1722 µg/mL, when compared to the vehicle control value.
In the presence of S9 mix following 3-hour treatment, 2-Methylene-1,3-propanediol diacetate caused no reduction in the mitotic index at 1722 µg/mL, when compared to the vehicle control value.
In the absence ofS9 mix following 21-hour continuous treatment, 2-Methylene-1,3prnpanediol diacetate caused a reduction in the mitotic index to 52% of the vehicle control value at 1722 µg/mL.
Therefore, the maximum final concentration selected for testing was 1722 µg/mL (1 OmM), the limit guideline concentration .. Concentiations used in the main test were based upon these data.

Main test - Treatment of cells with test substance
Vehicle control, treatment and positive control cultures were treated approximately 48 hours after commencement of incubation of lymphocyte cultures .. Duplicate cultures were prepared throughout for each treatment (3-hour treatment in the absence and presence of S9 mix, and 21-hour continuous treatment in the absence of S9 mix). All cultures were centrifuged and resuspended in the required volume of fresh medium before treatment, taking into account the treatment volume and S9 mix volume, where required,

In the absence of 59 mix, 3-hour treatment
2-Methylene-1,3-propanediol diacetate was added to each culture in 50 µL aliquots to give final concentrations of 223 17, 371.95, 619.92, 1033 20 and 1722 µg/mL DMSO was used as the vehicle control, and Mitomycin C at a final concentration of 0.2 µg/mL was the positive control.
After 3-hour treatment, no notable culture medium changes were observed, when compared to the vehicle control.
Following 3-hour treatment, cultures were centrifuged at 500g for 5 minutes and the supernatant removed. Cultures were then resuspended in saline (5 mL) and centrifuged at 500g for 5 minutes, The saline was then removed and the cell pellets resuspended in fresh medium. They were then incubated for a further 18 hours. The cultures were then harvested, slides prepared, and microscopically examined for cytotoxicity using a light microscope,

In the presence of 59 mix, 3-hour treatment
For treatments in the presence ofS9 mix, 1 mL ofS9 mix was added to give a concentration of 5% v/v in the final test medium
2-Methylene-1,3-propanediol diacetate was added to each culture in 50 µL aliquots to give final concentrations of 233.1 7, 371.95, 619.92, I 033.20 and 1722 µg/ml DMSO was used as the vehicle control, and Cyclophosphamide at a final concentration of 7..5 µg/mL was the positive control
After 3-hour treatment, no notable culture medium changes were observed, when compared to the vehicle control..
Following 3-hour treatment, cultures were centrifuged at 500g for 5 minutes and the supernatant removed. Cultures were then resuspended in saline (5 ml) and centrifuged at 500g for 5 minutes. The saline was then removed and the cell pellets resuspended in fresh medium They were then incubated for a further 18 hours. The cultures were then harvested, slides prepared, and microscopically examined for cytotoxicity using a light microscope
In the absence of S9 mix, 21-hour continuous treatment
2-Methylene-1,3-propanediol diacetate was added to each culture in 50 µL aliquots to give final concentrations of233 .17, 3 71.95, 619 .. 92, 1033 .20 and 1722 µg/mL. DMSO was used as the vehicle conttol, and Mitomycin C at a final concentration of 0. I µg/mL was the positive control.
After 21-hom treatment, no notable cultme medium changes were observed, when compared to the vehicle control. The cultures were then harvested, slides prepared, and microscopically examined for cytotoxicity using a light microscope.

Harvesting and fixation
Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid® to each culture at a final concentration of 0.1 µg/mL After 2 hours incubation, each cell suspension was transferred to a centrifuge tube and centrifuged for 5 minutes at 500g The cell pellets were treated with a hypotonic solution (0. 075M KCI), pre-warmed at 37°C. After a 10 minute period of incubation at 37°C, the suspensions were centrifuged at 500g for 5 minutes and the cell pellets fixed by addition of freshly prepared cold fixative (3 parts methanol : 1 part glacial acetic acid). The fixative was replaced until it was clear.

Slide preparation
The pellets were resuspended, then centrifuged at 500g for 5 minutes and finally resuspended in a small volume of fresh fixative. A few drops of the cell suspensions were dropped onto pre-cleaned microscope slides and allowed to air dry. The slides were then stained in 10% Giemsa, prepared in buffered water (pH 6.8). After rinsing in buffered water the slides were left to air-dry and mounted in DPX. The remainder of the cell pellets in fixative were stored at approximately 4°C until slide analysis was completed

Microscopic examination
The prepared slides were examined by light microscopy using a low power objective. The proportion of mitotic cells per 1000 cells in each culture was recorded (except for positive control treated cultutes, or in cultures where there were no signs ofcytotoxicity following assessment of sufficient concentrations to permit metaphase analysis)
From these results the concentration causing a decrease in mitotic index of at least 50% (where possible) of the vehicle control value was the highest concentration selected for metaphase analysis. Intermediate and low concentrations were also selected.
Where no biologically significant decrease in toxicity was observed (Le. no significant reduction in mitotic index greater than 50%), the maximum concentration tested (1722 µg/mL) was the highest concentration selected for metaphase analysis. Lower concentrations were also selected.
The selected slides were then coded. Metaphase cells were identified using a low power objective and examined at a magnification of x lOOO using an oil immersion objective One hundred metaphase figures were examined from each culture. Chromosome aberations were scored according to the classification of the ISCN (2009). Only cells with 44 - 48 chromosomes were analysed. The vernier readings of all aberant metaphase figures were recorded.
The incidence of polyploid and endoreduplicated cells (ie. the ploidy status) was recorded as a percentage of the I 00 metaphases analysed per slide.
The number o faberrant metaphase cells in each test substance group was compared with the vehicle control value using the one-tailed Fisher exact test (Fisher 1973).
A Cochran-Armitage test for trend (Armitage, 1955) was applied to the control and all test substance groups .. If this is significant at the 1% level, the test is reiterated excluding the highest concentration group - this process continues until the trend test is no longer significant
D20's (the minimum concentration (mg/mL) at which aberrations were found in 20% of metaphases) were estimated (where possible) using logistic regression on a log (concentration) scale, allowing the number of control aberrations to be non-zero (Armitage et aL, 2002). The following model was used

D20’s (the minimum concentration (mg/mL) at which aberrations were found in 20% of metaphases) were estimated (where possible) using logistic regression on a log(concentration) scale, allowing the number of control aberrations to be non-zero (Armitage et al., 2002). The following model was used:

p = C + (1 – C) / 1 + exp{– intercept – slope ln(conc)}

p is the proportion of cells with aberrations, conc is the concentration of test substance. C is a parameter estimating the control proportion of aberrations.

The D20 values for structural aberrations (including and excluding gaps) are retained within the study data.
The data was analysed using the SAFEStat (SAS statistical applications for end users, version 1.1) Chromosome Aberrations application (version 1.1) which was developed in SAS (SAS INSTITUTE 2002).

Stability, homogeneity and formulation analysis
The stability and homogeneity of the test substance and ofthe test substance in the vehicle were not deter mined as part of this study
Analysis of achieved concentration was not performed as part of this study

Evaluation criteria:

An assay was considered to be acceptable if the negative and positive control values lie within the current historical control range.
The test substance was considered to cause a positive response if the following conditions were met:
Statistically significant increases (p<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) were observed at one or more test concentration.
The increases exceed the vehicle control range of this laboratory, taken at the 99% confidence limit.
The increases were reproducible between replicate cultures.
The increases were not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
Evidence of a concentration-related response was considered to support the conclusion.
A negative response was claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any concentration.
A further evaluation would be carried out if the above criteria for a positive or a negative response are not met.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test
Toxicity data
In the absence of S9 mix following 3-hour treatment, 2-Methylene-1,3-propanediol diacetate caused no reduction in the mitotic index at 1722 µg/ml, the highest tested concentration, when compared to the vehicle control value.
In the presence of S9 mix following 3-hour treatment, 2-Methylene-1,3-propanediol diacetate caused no reduction in the mitotic index at 1722 µg/mL, the highest tested concentration, when compared to the vehicle control value
In the absence of S9 mix following 21-hour continuous treatment, 2-Methylene-1,3-propanediol diacetate caused a reduction in the mitotic index to 52% of the vehicle control value at 1722 µg/mL, the highest tested concentration.
The concentrations used in the main test were based upon these data

Main test: 3-hour treatment in the absence of S9 mix
Toxicity data
2-Methylene-l, 3-propanediol diacetate caused a reduction in the mitotic index to 84% of the vehicle control value at 1722 µg/ml. The concentrations selected for metaphase analysis were 619.92, 1033.20 and 1722 µg/mL

Metaphase analysis
2-Methylene-1,3-propanediol diacetate caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any analysed concentration, when compared to the vehicle control value.
All mean values for the vehicle control (DMSO), and all 2-Methylene-1,3-propanediol diacetate treatment concentrations were within laboratory historical control range, when taken at the 99% confidence limit
The positive control compound, Mitomycin C, caused statistically significant increases (p<0.001) in the proportion of aberrant cells. This demonstrated the sensitivity of the test system

Polyploidy and endoreduplication analysis
No statistically significant increases in polyploid or endoreduplicated metaphases were observed during metaphase analysis, when compared to the vehicle control.

Main test: 3-hour treatment in the presence of S9 mix
Toxicity data
2-Methylene-1,3-propanediol diacetate caused no reduction in the mitotic index at 1722 µg/ml, when compared to the vehicle control The concentrations selected for metaphase analysis were 619 .. 92, 1033 20 and 1722 µg/mL.

Metaphase analysis
2-Methylene-1,3-propanediol diacetate caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any analyzed concentration, when compared to the vehicle control value.
All mean values for the vehicle control (DMSO), and all 2-Methylene-1,3-propanediol diacetate treatment concentrations were within laboratory historical control range, when taken at the 99% confidence limit
The positive control compound, Cyclophosphamide, caused statistically significant increases (pIn the presence of S9 mix, 2-Methylene-1,3-propanediol diacetate did not demonstrate any evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system. As no increases in the mean values (both including and excluding gaps) were observed at any analysed concentration that exceeded the laboratory historical control range, within acceptable levels of toxicity, it was considered not to be beneficial to perform a direct repeat of the assay.

Polyploidy and endoreduplication analysis
No statistically significant increases in polyploid or endoreduplicated metaphases were observed during metaphase analysis, when compared to the vehicle control.

Main test: 21-hour continuous treatment in the absence of S9 mix
As 2-Methylene-l, 3-prnpanediol diacetate was negative for both short term treatments (3-hom treatment in the absence and presence of S9 mix), in accordance with current guidelines, cultures treated continuously for 21 hours in the absence ofS9 mix underwent metaphase analysis,

Toxicity data
2-Methylene-1,3-propanediol diacetate caused a reduction in the mitotic index to 67% of the vehicle control value at 1722 µg/ml, The concentrations selected for metaphase analysis were 37195, 1033.20 and 1722 µg/mL.

Metaphase analysis
In the absence of S9 mix following 21-hour continuous treatment, 2-Methylene-1,3 propanediol diacetate caused statistically significant increases at 1722 µg/ml (p<0.,001; including and excluding gaps), when compared to the vehicle control. At 1722 µg/mL, a cytotoxic concentration, mean values exceeded the laboratory historical control range when taken at the 99% confidence limit, with increases reproducible between duplicate cultures. No statistically significant increases were observed at the lower tested concentrations of 371 95 and 1033 20 µg/mL with mean values within the laboratory historical control range, when taken at the 99% confidence limit,
Mean values for the vehicle control (DMSO) were within the laboratory historical control range, when taken at the 99% confidence limit
The positive control compound, Mitomycin C, caused statistically significant increases (p<0.,001) in the proportion of aberrant cells This demonstrated the sensitivity of the test system

Polyploidy and endoreduplication analysis
No statistically significant increases in polyploid or endoreduplicated metaphases were observed during metaphase analysis, when compared to the vehicle control.

Table1           Summary of results

 

Exposure period	S9 mix	Nominal concentration of Test Substance	Cells with aberrations excluding gaps			Cells with aberrations including gaps			Relative Mitotic
(hours)	v/v	(µg/mL)	Individual values (%)		Mean (%)	Individual values (%)		Mean (%)	Index (%)
3	-	0 (DMSO)	2.0	5.0	3.5	4.0	6.0	5.0	100
		619.92	1.0	1.0	1.0	3.0	3.0	3.0	102
		1033.20	0.0	1.0	0.5	0.0	1.0	0.5	112
		1722	0.0	0.0	0.0	1.0	4.0	2.5	84
		0.2 (Mitomycin C)	18.0	18.0	18.0***	26.0	24.0	25.0***	-
3	+	0 (DMSO)	0.0	1.0	0.5	1.0	2.0	1.5	100
		619.92	0.0	4.0	2.0	1.0	4.0	2.5	94
		1033.20	0.0	2.0	1.0	2.0	2.0	2.0	109
		1722	2.0	1.0	1.5	4.0	2.0	3.0	102
		7.5 (Cyclophosphamide)	16.0	20.0	18.0***	18.0	22.0	20.0***	-
21	-	0 (DMSO)	0.0	1.0	0.5	1.0	1.0	1.0	100
		371.95	2.0	3.0	2.5	2.0	4.0	3.0	96
		1033.20	0.0	3.0	1.5	3.0	4.0	3.5	77
		1722	6.0	6.0	6.0***	10.0	10.0	10.0***	67
		0.1 (Mitomycin C)	34.0	25.0	29.5	41.0	27.0	34.0***	-

Test Substance: 2-Methylene-1,3-propanediol diacetate

One-tailed Fisher's exact test

***                    p<0.001

Otherwise         p>0.05

Conclusions:

It is concluded that 2-Methylene-1,3-propanediol diacetate has shown evidence of causing an increase in the frequency of structural chromosome aberrations, in the absence of S9 mix only following 21-hout continuous treatment, under the experimental conditions described

Executive summary:

This study was performed to assess the ability of 2-Methylene-1,3-propanediol diacetate to cause strnctmal chromosome aberrations in human lymphocytes cultured in vitro.

 

Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin (PHA), and exposed to the test substance both in the absence and presence of S9 mix derived fiom rat livers. Vehicle and positive control cultmes were also included . Two hours before the end of the incubation period, cell division was arrested using Colcemid®, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage

 

A preliminary toxicity test was performed in order to determine the toxicity of 2-Methylene-1,3-propanediol diacetate to cultured human lymphocytes A 3-hour treatment in the absence and presence of S9 mix, and a 21-hour continuous treatment in the absence of S9 mix were used to determine toxicity.

 

In the main test, the mitotic index was assessed for all cultures treated with 2-Methylene-1,3-propanediol diacetate and the vehicle control, dimethyl sulphoxide (DMSO) Justification for the highest analysed concentration was based on the limit final concentration (I 0 mM) for this test system.

 

On the basis of these data, the following concentrations were selected for metaphase analysis:

Tn the absence of S9 mix, 3-hour treatment: 619.92, 103.3.20 and 1722 µg/mL.

Tn the presence of S9 mix, 3-hour treatment: 619 .92, 1033 .20 and 1722 µg/mL.

In the absence of S9 mix, 21-hour continuous treatment: 371 .95, 1033 .20 and 1722 µg/mL.

 

In the absence and presence of S9 mix following 3-hour treatment, 2-Methylene-1,3-propanediol diacetate caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any analysed concentration, when compared to the vehicle control Mean values for the vehicle control (DMSO) and all treatment concentrations were within the laboratmy historical control range, when taken at the 99% confidence limit.

 

In the absence of S9 mix following 21-hour continuous treatment, 2-Methylene-1,3-propanediol diacetate caused statistically significant increases (p<0.001: including and excluding gaps) in the proportion of metaphase figures containing chromosomal aberrations, at 1722 µg/mL (a cytotoxic concentration), when compared to the vehicle control At 1722 µg/mL, mean values exceeded the laboratory historical control range when taken at the 99% confidence limit. No statistically significant increases were observed at the lower tested concentrations of 371.95 and 1033.20 µg/ml, with mean values within the laboratory historical control range, when taken at the 99% confidence limit. Mean values for the vehicle control (DMSO) were within the laboratory historical control range, when taken at the 99% confidence limit

 

No statistically significant increases in the proportion of polyploid or endoreduplicated metaphase cells were observed during metaphase analysis, under any treatment condition, when compared to the vehicle control.

 

Both positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

It is concluded that 2-Methylene-1,3-propanediol diacetate has shown evidence of causing an increase in the frequency of structmal chromosome aberations, in the absence of S9 mix only following 21-hour continuous treatment, under the experimental conditions described

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In the CD1 Mouse In Vivo Comet Test including Micronucleus Analysis (Huntingdon Life Sciences, 2014, LYA0043) it was concluded that the test item 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.

It the micronucleus Phase the test item has not shown any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male

CDI mice when administrated orally by gavage in this in vivo test procedure.

Link to relevant study records

Reference

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

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

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

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EC No. 440/2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

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

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

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.

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

Dose / conc.:

62.5 other: mg/kg/day

Dose / conc.:

125 other: mg/kg/day

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

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

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)