Registration Dossier

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:
From 2012-11-02 to 2012-11-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Aminopropyl Vinyl Ether
- Test-substance No.: 12/0567-1
- Lot/batch No.: C520/070/2012/2
- Purity/composition: Water content: 99.6 corrected area-%
- Homogeneity: The homogeneity of the test substance was guaranteed by mixing before preparation of the test substance solutions
- Storage stability: The stability of the test substance under storage conditions throughout the study period was guaranteed until 27 Jun 2016 as indicated by the sponsor, and the sponsor holds this responsibility
- Physical state: Liquid, colourless, clear
- Storage conditions: Room temperature, avoid temperatures >25°C

Method

Target gene:
- Salmonella typhimurium: histidine
- Escherichia coli: tryptophan
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/ß-naphthoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
0; 33; 100; 333; 1000; 2500 and 5200 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: Ultrapure water
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in ultrapure water, ultrapure water was used
as vehicle.
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9 mix for all strains
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-Methyl-N'-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix for strains TA 1535 and TA 100
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine
Remarks:
Without S9 mix for strain TA 98
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without S9 mix for strain TA 1537
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without S9 mix for strain E. coli WP2 uvrA
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
Standard plate test
- Exposure duration: 48 - 72 hours at 37°C in the dark
Preincubation test
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 48 - 72 hours at 37°C in the dark

NUMBER OF REPLICATIONS: 3 test plates per dose or per control
Evaluation criteria:
Toxicity detected by a decrease in the number of revertants , clearing or diminution of the background lawn (= reduced his- or trp- background growth), reduction in the titer is recorded for all test groups both with and without S9 mix in all experiments and indicated in the tables.
Acceptance criteria
Generally, the experiment is considered valid if the following criteria are met: The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. The sterility controls revealed no indication of bacterial contamination. The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above. Fresh bacterial culture containing approximately 109 cells per mL were used. For approval the titer of viable bacteria was ≥ 108 colonies per mL.
Assessment criteria
The test substance is considered positive in this assay if the following criteria are met: A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system. A test substance is generally considered non-mutagenic in this test if: The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
from about 2600 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TOXICITY
A bacteriotoxic effect decrease in the number of his+ or trp+ revertants, slight reduction in the titer) was occasionally observed in the standard plate test depending on the strain and test conditions from about 2 600 μg/plate onward. In the preincubation assay bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed depending on the strain and test conditions from about 2 600 μg/plate onward.

SOLUBILITY
No test substance precipitation was found neither with nor without S9 mix.

DISCUSSION
According to the results of the present study, the test substance did not lead to an increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in three experiments carried out independently of each other (standard plate test and preincubation assay).
Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.

Applicant's summary and conclusion

Conclusions:
Thus, under the experimental conditions chosen here, it is concluded that Aminopropyl Vinyl Ether is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.