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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:
Sep 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and batch No.of test material: BASF SE and A16377
- Date of production: Dec 2016
- Purity: 96.53% (the exact value is currently being analyzed)
- Appearance: liquid, colorless, clear

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator
- Stability under test conditions: The stability of the test substance under storage conditions is guaranteed until Dec 2018
- Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substanc solution

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was weighed and topped up with DMSO to achieve the required concentration of the stock solution.
- Final dilution of a dissolved stock liquid: Further concentrations were diluted from the stock solution according to the plannes doses

OTHER SPECIFICS:
- All test substance formulations were prepared immediately before administration

Method

Target gene:
His G 46, his C 3076, his D 3052, and trp
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction derived from phenobarbital and beta-naphthoflavone induced rat liver
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction derived from phenobarbital and beta-naphthoflavone induced rat liver
Test concentrations with justification for top dose:
1st experiment: 33, 100, 333, 1000, 2600 and 5200 µg/plate (all strains in standard plate test)
2nd experiment: 3.3, 10, 33, 100, 333, and 1000 µg/plate (all strains in standard plate test)
3rd experiment: 1, 3.3, 10, 33, 100, and 333 µg/plate (TA strains) (preincubation test)
3.3, 10, 33, 100, 333, and 1000 µg/plate (E.Coli WP2 uvrA) (preincubation test)

In agreement with the recommendations of current guidelines 5 mg/plate were generally selected as maximum test dose at least in the 1st experiment. However, this maximum dose was tested even int he case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeated experiments for further clarification/substantiation. In this study, due to the purity of the test substance 5.2 mg/plate was used as top dose in the 1st experiment.
Vehicle:
- Vehicle: DMSO
- Justification for choice of vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Controls
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: 2-aminoanthracene 2.5 µg/plate dissolved in DMSO strins TA 1535, TA 100, TA 1537, and TA 98; N-methyl-N'-nitro-N-nitrosoguanidine 5 µg/plate dissolved in DMSO strains TA 1535, TA 100; 4-nitro-o-phenylenediamine 10 µg/plate dissolved in DMSO strain TA 98
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION OF STANDARD PLATE TEST
- Exposure duration: 48 - 72 h in the dark

DURATION OF PREINCUBATION TEST
- Preincubation time: 20 minutes
- Exposure duration: 48 - 72 h in the dark

NUMBER OF REPLICATIONS: 3 test plates per dose/control

DETERMINATION OF CYTOTOXICITY
- Method: Decrease in the number of revertants and clearing or diminution of the background lawn (= reduced his- or trp- background growth)

DETERMINATION OF MUTAGENICITY
- Method: Individual plate counts, the mean number of revertant colonies per plate and the standard deviation

Evaluation criteria:
ASSESSMENT CRITERIA
The test substance was considered positive in this assay if the following critera were met:
- a dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) 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 considered non-mutagenic in this test if:
- the number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

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 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 10^9 cells per ml were used

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
from about 100 µg/plate onward
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
from about 100 µg/plate onward
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
from about 1000 µg/plate onward
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
from about 333 µg/plate onward
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
from about 333 µg/plate onward
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no test substance precipitation was found with and without S9 mix

HISTORICAL CONTROL DATA (with ranges, means and standard deviation)
- Positive historical control data: see attached background material
- Negative vehicle historical control data: see attached background material

Applicant's summary and conclusion

Conclusions:
In conclusion, based on the experimental conditions chosen the test substance is not a mutagene in the bacterial reverse mutation test in the absence and in 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 several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

DOSE RANGE: 3.3 μg - 5200 μg/plate (SPT)

1.0 μg - 1000 μg/plate (PIT)

TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

SOLUBILITY: No precipitation of the test substance was found with and without S9 mix.

TOXICITY: A bacteriotoxic effect was observed depending on the strain and test conditions from about 100 μg/plate onward

MUTAGENICITY: A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.