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Genetic toxicity: in vitro

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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471, "Bacterial Reverse Mutation Test", adopted 21st July, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Commission Regulation (EC) No. 440/2008 B.13/14:"Mutagenicity - Reverse Mutation Test using Bacteria", dated May 30, 2008.
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Methacrylic anhydride
EC Number:
212-084-8
EC Name:
Methacrylic anhydride
Cas Number:
760-93-0
Molecular formula:
C8H10O3
IUPAC Name:
2-methylacrylic anhydride
Test material form:
liquid

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: his D 3052; rfa-; uvrB-; R-factor: frame shift mutations
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: his G 46; rfa-; UV1-B"; R-factor: base-pair substitutions
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
other: his G 46; rfa-; UV1-B": base-pair substitutions
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
other: his C 3076; rfa-; uvrB-: frame shift mutations
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: his G 428 (pAQI); rfa-; R-factor: base-pair substitutions
Test concentrations with justification for top dose:
The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment .
2.5 µL/plate was selected as the maximum concentration, The concentration range covered two logarithmic decades. Two independent experiments were performed with the following concentrations:

Experiment I:
0.00100,0.00316,0.0100,0.0316,0.100,0.316 and 1.0 µL/plate (TA 1535, TA 1537 and TA 102 without metabolic activation)
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0 and 2.5 µL/plate
(with metabolic activation and TA 98, TA 100 without metabolic activation)
Experiment II:
0.00200, 0.00632, 0.0200, 0.0632, 0.200, 0.632 and 1.0 µL/plate
(without metabolic activation)
0.00632, 0.0200, 0.0632, 0.200, 0.632, 1.0 and 2.0 µL/plate
(with metabolic activation)
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
Preparation of Bacteria
SampIes of each tester strain were grown by culturing for 12 h at 38,5 °C in Nutrient Broth to the late exponential or early stationary phase of growth (approx, 10E+9 cells/mL),
The nutrient medium consists per litre:
8 g Nutrient Broth
5 g NaCI
A solution of 125 µL ampicillin (10 mg/mL) (TA 98, TA 100, TA 102) was added in order to retain the phenotypic characteristics of the strain,

Agar Plates
The Vogel-Bonner Medium E agar plates with 2% glucose used in the Ames Test were prepared by BSL BIOSERVICE GmbH or provided by an
appropriate supplier. Quality controls were performed.
Vogel-Bonner-salts contain per litre:
10 g MgS04x 7H2O
100 g citric acid
175 g NaNH4HP04 x 4 H2O
500 g K2HP04

Sterilisation was performed at 121°C in an autoclave.
Vogel-Bonner Medium E agar plates contain per litre:
15 g Agar Agar
20 mL Vogel-Bonner salts
50 mL glucose-solvent (40%)
Sterilisation was performed at 121°C in an autoclave.

Overlay Agar
The overlay agar contains per litre:
7.0 g Agar Agar
6.0 g NaCI
10.5 mg L-histidine x HCl x HzO
12.2 mg biotin
Sterilisation was performed at 121°C in an autoclave.

Mammalian Microsomal Fraction S9 Mix
The bacteria most commonly used in these reverse mutation assays do not possess the enzyme system which, in mammals, is known to convert
promutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous
metabolic system was added in form of mammalian microsome enzyme activation mixture.

S9 Homogenate
The S9 liver microsomal fraction was preparcd at BSL BIO SERVICE GmbH. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and
ß-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route.
The following quality control determinations are performed:
a) Biological activity in:
- the Salmonella typhimurium assay using 2-aminoanthracene
-the mouse lymphoma assay using benzol [ a ]pyrene
-the chromosome aberration assay using cyclophosphamide.
b) Sterility Test
A stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4.5 mL and stored at <-75 °C.
The protein concentration in the S9 preparation (Lot: 110310) was 31 mg/mL. The S9 mix preparation was performed according to Ames et al.
Preparation of S9 Mix
100 mM of ice-cold sodium-Ortho-phosphat-buffer, pH 7.4, was added to the following pre-weighed sterilised reagents to give final concentrations in the S9 mix of:
8 mM MgCl2; 33 mM KCI; 5 mM glucose-6-phosphate; 4 mM NADP
This solution was mixed with the liver 9000 x g supernatant fluid in the following proportion:
co-factor solution 9.5 parts
liver preparation 0.5 parts
During the experiment the S9 mix was stored on ice.

S9 Mix Substitution Buffer
The S9 mix substitution buffer was used in the study as a replacement of S9 mix,
without metabolic activation (-S9).
Phosphate-buffer (0.2 M) contains per litre:
0.2 M NaH2P04 x H20: 120 mL
0.2 M Na2HP04: 880 mL
The two solutions were mixed and the pH was adjusted to 7.4. Sterilisation was performed at 121°C in an autoclave.
This 0.2 M phosphate-buffer was mixed with 0.15 M KCI solution (sterile) in the following proportion:
0,2 M phosphate-buffer: 9.5 parts
0.15 M KCI solution: 0.5 parts
This S9 mix substitution buffer was stored at 4°C.

Experimental Performace
For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL: Test solution at eaeh dose level, solvent control, negative control of reference mutagen solution (positive control),
500 µL: S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 µL: Bacteria suspension (cf. Preparation of Baeteria, pre-culture of the strain),
2000 µL: Overlay agar

For each strain and dose level, including the controls, three plates were used,
After solidification the plates were inverted and incubated at 37°C for at least 48 h in the dark.

Data Recording
The colonies were counted using a ProtocoL counter (Meintrup DWS Laborgeräte GmbH). If precipitation of the test item precluded automatic
counting the revertant colonies were counted by hand, in addition, tester strains with a low spontaneous mutation frequency like TA 1535 and TA 1537 were counted manually,
Evaluation criteria:
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn
or a reduction in the number of revertants down to
a mutation factor of approximately <= 0.5 in relation to the solvent control

A test is considered acceptable if for each strain: the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100, TA 102) the control plates with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data
range):
- S9 +S9
TA 98: 18 18-46 18- 57
TA 100: 77 - 163 78 - 165
TA 1535: 5 -29 5-27
TA 1537: 5 -30 5 -36
TA 102: 164 -390 163 -472
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control Plate

The Mutation Factor is calculated by dividing the mean value of the revertant counts
through the mean values of the solvent control
A test item is considered as mutagenic if:
a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.

Acc. to OECD guidelines, the biol. relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
It can be stated that during the described mutagenicity test and under the experimental conditions reported, Methacrylic anhydride did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, Methacrylic anhydride is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

In a reverse gene mutation assay in bacteria (Ames test), strains TA1535, TA1537, TA98, TA100, and TA102 of Salmonella typhimuriumwere exposed to Methacrylic anhydride at concentrations of up to 2.0 µl/plate in the presence and absence of mammalian metabolic activation S9 -mix. 

No biologically relevant inereases in revertant colony numbers of any of the five tester strains were observed following treatment with Methacrylic anhydride at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II

The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Methacrylic anhydride did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.

Therefore, Methacrylic anhydride is considered to be non-mutagenic in this bacterial reverse mutation assay. Appropriate reference mutagens were used as positive controls.

The positive controls induced the appropriate responses in the corresponding strains.

This study is classified as acceptable. This study satisfies the requirement for tests for in vitro mutagenicity (bacterial reverse gene mutation) data.

This study is classified as acceptable, it satisfies the requirements for testing bacterial reverse mutations.

 

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

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