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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:
06 Dec 2016 - 15 Dec 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Qualifier:
according to
Guideline:
EPA OTS 798.5100 (Escherichia coli WP2 and WP2 UVRA Reverse Mutation Test)
Version / remarks:
1998
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment. No bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test up to the highest concentration.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, ethanol 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
Untreated negative controls:
yes
Remarks:
Additional plates were treated with soft agar, S9 mix, buffer, vehicle and the test substance but without the addition of tester strains (sterility control)
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation

DURATION
- Exposure duration: 48 – 72 hours

NUMBER OF REPLICATIONS: 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- decrease in the number of revertants (factor ≤ 0.6)
- clearing or diminution of the background lawn (= reduced his- or trp- background growth)

POSITIVE CONTROLS
With S9 mix
• 2-aminoanthracene (2-AA)
- 2.5 μg/plate, dissolved in DMSO, strains: TA 1535, TA 100, TA 1537, TA 98
- 60 μg/plate, dissolved in DMSO, strain: Escherichia coli WP2 uvrA
Without S9 mix
• N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
- 5 μg/plate, dissolved in DMSO, strains: TA 1535, TA 100
• 4-nitro-o-phenylenediamine (NOPD),
- 10 μg/plate, dissolved in DMSO, strain: TA 98
• 9-aminoacridine (AAC)
- 100 μg/plate, dissolved in DMSO, strain: TA 1537
• 4-nitroquinoline-N-oxide (4-NQO)
- 5 μg/plate, dissolved in DMSO, strain: E. coli WP2 uvrA
Evaluation 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 (bacteria strains with high spontaneous mutation rate, like TA 98, 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 was generally 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.

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from about 333 μg/plate onward in the standard plate test and from about 1000 μg/plate in the preincubation test both with and without S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test up to the highest concentration. In the preincubation assay bacteriotoxicity (reduced his- background growth, decrease in the number of his+ revertants) was observed using the tester strain TA 1535 and TA 1537 without S9 mix at a concentration of 5000 μg/plate.

Any other information on results incl. tables

SUMMARY OF RESULTS

Standard Plate Test:

Mean revertants per plate
Strain TA 1535 TA 100 TA 1537 TA 98 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Ethanol 10.3 14.0 85.3 102.7 6.0 9.3 26.0 31.3 20.0 18.3
33 12.0 9.3 89.3 100.7 8.0 7.3 23.3 26.7 16.7 25.3
100 11.3 13.3 82.3 92.0 7.7 9.7 24.7 23.7 14.7 19.3
333 10.7 14.0 94.0 92.0 7.3 10.0 20.0 30.0 18.0 20.3
1000 9.7 10.3 77.3 93.7 7.3 7.0 18.7 35.0 15.3 30.0
2500 12.7 10.0 72.7 92.3 11.0 7.7 22.7 43.3 14.7 24.0
5000 10.0 10.0 76.3 78.3 4.0 9.3 18.3 22.3 19.3 19.0
positive control 5005.7 210.7 2840.0 2030.7 1252.7 154.3 585.7 1914.3 1570.3 117.0

Preincubation Test:

Mean revertants per plate
Strain TA 1535 TA 100 TA 1537 TA 98 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Ethanol 10.3 9.7 78.3 108.0 5.3 7.0 20.3 34.3 27.7 27.3
33 15.3 15.7 88.7 107.0 6.3 6.3 23.3 30.0 22.3 33.3
100 6.7 6.7 93.7 99.3 7.7 7.0 19.7 36.7 28.7 21.7
333 13.7 9.0 88.0 101.0 6.7 9.3 20.0 33.3 24.3 27.3
1000 12.3 10.3 110.3 106.3 7.0 6.3 16.0 25.3 20.3 24.0
2500 10.7 8.7 90.3 81.3 6.7 4.7 29.3 26.3 21.3 29.7
5000 6.0 10.3 85.0 81.7 0.3 10.0 23.3 29.0 25.0 19.3
positive control 2743.0 215.7 2331.7 2443.7 1058.0 144.3 542.3 2177.0 311.7 102.7

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/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 several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. The strains used were TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA with a dose range of 33 μg - 5000 μg/plate in both standard plate test (SPT) and preincubation test (PIT) each performed with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was found from about 333 μg/plate onward with and without S9 mix. A bacteriotoxic effect was occasionally observed depending on the strain and test conditions at a concentration of 5000 μg/plate. 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. Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.