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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22.10.2009 to 15.12.2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
solid
Specific details on test material used for the study:
4-(Methacryloyloxy) bezophenone 97.26 %
Benzophenone acetate: 1.95 %

Method

Target gene:
his-locus
TA98: his D 3052; rfa"; uvrB·; R-factor: frame shift mutations
TA 100: his G 46; rfa·; uvrB"; R-factor: base-pair substitutions
TA 1535: his G 46; rfa·; uvrB": base-pair substitutions
TA 1537: his C 3076; rfa"; uvrB·: frame shift mutations
TA 102: his G 428 (pAQI ); rfa"; R-factor: base-pair substitutions
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiment I:
10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
3.16, 10.0, 31.6, 100, 316, 1000 and 2500 μg/plate (only TA I 02 without metabolic activation)
Experiment II:
3.16, 10.0,31.6, 100,316, 1000 and 2500 μg/plate (TA 98 and TA 1535 with and without metabolic activation)
1.00, 3.16, 10.0, 31.6, 100, 316 and 1000 μg/plate (TA 100 and TA 1537 with and without metabolic activation, TA 102 with metabolic activation)
0.316, 1.00, 3 .16, 10.0, 31.6, 100 and 316 μg/plate (TA 102 without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties + relative nontoxicity to the bacteria
Controlsopen allclose all
Positive controls:
yes
Remarks:
without metablic activation
Positive control substance:
sodium azide
Remarks:
Tester Strains: S. typhimurium: TA 100, TA 1535 Name: Sodium azide, NaN3 Purity: at least 99% Dissolved in: Aqua dest. Concentration: 10 µg/plate
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
other: 4-nitro-o-phenylene-diamine, 4-NOPD
Remarks:
Tester Strains: S. typhimurium: TA 98, TA 1537 Name: 4-nitro-o-phenylene-diamine, 4-NOPD Purity: >97% Dissolved in: DMSO Concentrations: 10 µg/plate forTA 98, 40 µ/plate for TA 1537
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
methylmethanesulfonate
Remarks:
Tester Strain: S. typhimurium: TA 102 Name: Methyl methane sulfonate, MMS Purity: 99.0% Dissolved in: Aqua dest. Concentration: 1 µg/plate
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
other: 2-aminoanthracene, 2-AA
Remarks:
Tester Strains: S. typhimurium: TA 98, TA 100, TA 1535, TA 1537 and TA 102 Purity: 96% Dissolved in: DMSO Concentrations: 2.5 µg/plate; 10 µg/plate for TA 102
Negative solvent / vehicle controls:
yes
Remarks:
Aqua dest.
Remarks:
Solvent controls, consisting of solvent or vehicle alone as well as untreated controls were treated in the same way as the treatment groups.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Remarks:
Solvent controls, consisting of solvent or vehicle alone as well as untreated controls were treated in the same way as the treatment groups.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 60 minutes
- Exposure duration: at least 48 h

NUMBER OF EXPERIMENTS: 2 (one performed as pre-incubation assay, one as plate incorporation assay), 3 plates per concentration each

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test article may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.

OTHER:
automatic colony count with “AUTOCOUNT” (Artek systems, Biosys)
Evaluation criteria:
- corresponding background growth on negative control and test plates
- normal range of spontaneous revertants: TA1535 (10-29), TA 1537 (5-28), TA 1538 (12-37), TA 98 (15-57), TA 100 (77-189)
- test material is considered mutagenic if either a dose related and reproducible increase in revertant numbers or a significant and reproducible increase in revertant numbers for at least one test concentration in induced
- a significant increase in revertants means at least twice as high in TA 100 and a three times higher number in TA 1535, TA 1537, TA 1538, TA 89 compared to the negative control

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: TA98, TA 100, TA 1535, TA 1537, 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

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, 4-(Methacryloyloxy) benzophenone did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, 4-(Methacryloyloxy) benzophenone is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471, adopted 21 July 1997, strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 were exposed to 4 -(Methacryloyloxy) benzophenone in DMSO at concentrations of 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000.0 µg/plate in a plate incorporation assay in the presence and absence of mammalian metabolic activation (S9 mix).

4 -(Methacryloyloxy) benzophenone was tested up to cytotoxic concentrations. There was no evidence of induced mutant colonies over background.

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

This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.