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Toxicological information

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
Study period:
04 Feb 2014 - 21 Feb 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
yes
Remarks:
only E. coli strain tested
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
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

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): 1,2-Propylendiamin techn.
- Physical state: colorless clear liquid
- Analytical purity: 99.7 %
- Lot/batch No.: 000STD77L0
- Storage condition of test material: room temperature
- Stability: The stability of the test substance under storage conditions throughout the study period was guaranteed by the manufacturer until 04 Nov 2015; the manufacturer holds this responsibility

Method

Target gene:
trp operon for the E. coli strain
Species / strain
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 liver mix prepared from Wistar rats treated with 80 mg/kg bw phenobarbital i.p. and β-naphthoflavone orally, each on three consecutive days.
Test concentrations with justification for top dose:
First experiment (standard plate test, with and without metabolic activation, 3 plates/dose or control): 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Second experiment (preincubation test with and without metabolic activation, 3 plates/dose or control): 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: due to the limited solubility of the test substance in water, DMSO was used as vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
with S9-mix
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
60 µg/plate in DMSO
Positive controls:
yes
Remarks:
without S9-mix
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
5 µg/plate in DMSO
Details on test system and experimental conditions:
STANDARD PLATE TEST (SPT)
According to Ames et al., Mut Res 31: 347-364 (1975) and Maron & Ames, Mut Res 113: 173-215 (1983)

In the standard plate test, tubes were filled with 2mL portions of soft agar and kept in a water bath at 42 to 45°C. This soft agar consisted of 100 mL agar and 10 mL amino acid solution.
Then following components are added:
0.1 mL test solution or vehicle
0.1 mL fresh bacterial culture
0.5 mL S9 -mix or phosphate buffer
After mixing samples were poured onto Merckoplate® (minimal glucose agar plates) plate and incubated for 48 - 72 hrs in the dark at 37°C.

PREINCUBATION TEST (PIT)
According to Yahagi et al. Mut Res 48: 121-129 (1977) and Matsushima et al., In: Norpoth, K.H. and R.C. Garner, Short-Term Test Systems for Detecting Carcinogens, Springer Verlag Berlin, Heidelberg, New York (1980)

For the preincubation test 0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL of either S9 mix or phosphate buffer were incubated at 37°C for 20 minutes. After addition of 2 mL soft agar, samples were poured onto agar plates and incubated again at 37°C for 48 to 72 hrs.

Evaluation criteria:
Acceptance criteria
Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the
historical negative control data for the tester strain.
• The sterility controls revealed no indication of bacterial contamination (see Appendix 3).
• 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.

Assessment criteria
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least
doubling of the spontaneous mutation rate in at least in the tester strain either without
S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for the tester strains were within the historical negative control
data range under all experimental conditions in at least two experiments carried out
independently of each other.

Results and discussion

Test results
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
no increase in number of revertants was observed
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from about 2500 µg/plate onward
Vehicle controls validity:
valid
Untreated negative controls validity:
other: sterility control, yes
Positive controls validity:
valid
Additional information on results:
A biologically relevant increase in the number of trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.
No bacteriotoxic effect was observed in the standard plate test.
In the preincubation assay bacteriotoxicity (reduced trp- background growth, slight decrease in the number of trp+ revertants) was occasionally observed depending on the test conditions from about 2 500 μg/plate onward.

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of this study, the test substance is not mutagenic in the Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.
Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of Escherichia coli WP2 uvrA, in a reverse mutation assay. The test concentrations were 0, 33, 100, 333, 1000, 2500, and 5000 µg/plate for the standard plate test with and without S9 mix, and for the preincubation test with and without S9 mix, respectively. Negative (sterility and solvent) and positive controls were considered.

Precipitation of the test substance was found from about 2 500 μg/plate onward with and without S9 mix. A weak bacteriotoxic effect was occasionally observed depending on the test conditions from about 2 500 μg/plate

onward.

A biologically relevant increase in the number of trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.