2-methylenepropane-1,3-diyl diacetate

Genetic toxicity: in vitro

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EC No. 440/2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

Test guideline 4

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

Test guideline 5

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

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-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

Method

Target gene:

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

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain 2

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.

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Any other information on results incl. tables

 

 

Applicant's summary and conclusion

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