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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test article was nut mutagenic in an Ames test.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 Feb 2017 - 16 Feb 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
his, trp
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:
liver S9 mix from induced rats
Test concentrations with justification for top dose:
0; 33; 100; 333; 1000; 3000 and 6000 μg/plate
Due to the purity of the test substance 6.0 mg/plate was used as top dose in all experiments.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO;
- Justification for choice of solvent/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
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 – 72 hours in the dark

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

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.
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:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test substance precipitation was found with and without S9 mix.

HISTORICAL CONTROL DATA
The number of revertant colonies in the negative controls with and without S9 was within the range of the historical negative control data for each tester strain. In addition, the positive control substances with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was observed in the standard plate test only with S9 mix using the tester strains TA 1537 from about 3000 μg/plate onward and TA 98 at a concentration of 6000 μg/plate. In the preincubation assay bacteriotoxicity (slight decrease in the number of his+ revertants) was observed using TA 1537 without S9 mix at a concentration of 6000 μg/plate.

EXPERIMENTAL RESULT

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
DMSO 10.7 10.3 96.0 106.7 7.7 12.0 21.3 31.3 23.3 21.0
33 10.3 8.3 108.3 111.7 10.3 12.0 16.7 25.7 27.0 26.0
100 11.7 10.3 113.7 118.7 7.3 9.7 17.7 34.7 20.3 23.7
333 13.7 12.7 100.3 118.7 10.3 9.3 16.0 19.3 22.7 29.3
1000 13.0 13.0 106.0 104.0 10.7 9.7 24.3 27.7 23.3 22.0
3000 14.0 14.0 115.3 107.3 7.7 7.0 21.7 21.3 22.7 28.3
6000 13.7 14.7 136.0 115.0 9.7 7.0 22.3 20.3 26.7 21.7
positive control 5382.7 188.7 4366.0 1330.3 1210.7 123.3 656.0 2513.0 1026.3 116.0

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
DMSO 7.7 10.3 80.3 94.7 6.7 8.7 20.0 32.3 23.0 23.7
33 10.7 5.0 86.0 86.0 6.3 11.0 18.3 36.7 30.3 18.7
100 8.7 9.3 94.3 85.0 7.0 8.7 19.0 23.7 23.7 17.3
333 9.7 9.3 86.0 89.0 7.3 11.3 20.0 31.3 16.7 24.0
1000 14.0 8.3 94.7 98.0 9.7 6.3 23.3 28.0 15.0 19.0
2600 14.3 13.0 85.7 103.3 6.7 11.0 15.7 27.0 20.7 24.3
5200 16.7 16.3 100.7 112.3 3.7 5.7 15.3 21.3 22.7 17.7
positive control 3495.3 228.3 2569.7 2330.7 1062.7 146.0 593.0 1850.0 606.3 103.0
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 TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA. The dose range tested was 33 μg - 6000 μg/plate both in the standard plate test and in the preincubation assay both with and without metabolic activation (liver S9 mix from induced rats). No precipitation of the test substance was found with and without S9 mix. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 3000 μg/plate onward. A biologically 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. Thus, 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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

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 TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA. The test was performed according to OECD guideline 471 and under GLP. The dose range tested was 33 μg - 6000 μg/plate both in the standard plate test and in the preincubation assay both with and without metabolic activation (liver S9 mix from induced rats). A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was observed in the standard plate test only with S9 mix using the tester strains TA 1537 from about 3000 μg/plate onward and TA 98 at a concentration of 6000 μg/plate. In the preincubation assay bacteriotoxicity (slight decrease in the number of his+ revertants) was observed using TA 1537 without S9 mix at a concentration of 6000 μg/plate. The test substance did not lead to a biologically relevant increase in the number of revertant colonies without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay). 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. 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 with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data. In conclusion, under the experimental conditions chosen here, it is concluded that is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.

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