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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:
2016-2017
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(21 Jul 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:
The test item was homogeneous by visual inspection.
Storage conditions: Room temperature
Batch No.: VFH-2016-08

Method

Target gene:
his+ / trp+
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and beta-naphthoflavone induced rat liver S9 microsomal fraction
Test concentrations with justification for top dose:
1st Experiment (Standard plate test with and without S9 mix): 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
2nd Experiment (Preincubation test with and without S9 mix): 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD), 9-aminoacridine (AAC), 4-nitroquinoline-N-oxide (4-NQO)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

Standard plate test:
- Exposure duration: 48 – 72 hours

Preincubation test:
- Preincubation period: 20 min
- Exposure duration: 48 – 72 hours


NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn, reduction in the titer


OTHER:
Titer determination: The titer was determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.

Positive controls:

With S9 mix: 2-aminoanthracene (2-AA), 2.5 μg/plate, dissolved in DMSO / TA 1535, TA 100, TA 1537, TA 98; 60 μg/plate, dissolved in DMSO / Escherichia coli WP2 uvrA
Without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 5 μg/plate, dissolved in DMSO / TA 1535, TA 100; 4-nitro-o-phenylenediamine (NOPD), 10 μg/plate, dissolved in DMSO / TA 98; 9-aminoacridine (AAC), 100 μg/plate, dissolved in DMSO / TA 1537; 4-nitroquinoline-N-oxide (4-NQO) (SIGMA, N-8141), 5 μg/plate, dissolved in DMSO / E. coli WP2 uvrA
Evaluation criteria:
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 10^9 cells per mL were used. For approval the titer of viable bacteria was ≥ 10^8 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 resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:A weak bacteriotoxic effect (slight decrease in the number of his+ or trp+ revertants) was occasionally observed in the standard plate and preincubation test from about 2500 μg/plate onward.

Any other information on results incl. tables

1st experiment, standard plate incorporation assay without metabolic activation
Strain test group dose (mg/plate) mean revertants per plate standard deviation factor individual colony count
TA 1535 DMSO - 11.3 4.0 - 7, 12, 15
Test item 33 11.7 2.5 1.0 14, 12, 9
100 6.7 3.1 0.6 6, 4, 10
333 10.7 4.0 0.9 10, 15, 7
1000 11.0 3.6 1.0 7, 14, 12
2500 10.7 3.5 0.9 14, 7, 11
5000 10.0 4.6 0.9 5, 14, 11
  MNNG 5.0 5591.7 410.3 493.4 6037, 5509, 5229
TA 100 DMSO - 112.0 14.9 - 118, 95, 123
Test item 33 103.7 9.3 0.9 96, 101, 114
100 109.7 6.5 1.0 103, 110, 116
333 104.0 11.0 0.9 104, 93, 115
1000 111.7 9.1 1.0 122, 105, 108
2500 109.3 5.0 1.0 114, 104, 110
5000 96.7 4.2 0.9 100, 92, 98
  MNNG 5.0 3747.3 403.7 33.5 4168, 3363, 3711
TA 1537 DMSO - 11.0 2.0 - 11, 9, 13
Test item 33 11.3 4.0 1.0 15, 7, 12
100 12.0 3.6 1.1 15, 8, 13
333 12.0 0.0 1.1 12, 12, 12
1000 8.3 3.5 0.8 8, 5, 12
2500 8.7 3.5 0.8 12, 5, 9
5000 11.7 2.5 1.1 9, 12, 14
  AAC 100 949.0 104.8 86.3 885, 1070, 892
TA 98 DMSO - 20.3 5.8 - 17, 27, 17
Test item 33 17.7 6.5 0.9 11, 18, 24
100 18.0 2.0 0.9 18, 16, 20
333 17.0 6.2 0.8 24, 12, 15
1000 17.0 1.7 0.8 16, 16, 19
2500 18.0 6.0 0.9 12, 24, 18
5000 19.0 7.2 0.9 27, 13, 17
  NOPD 10 591.3 9.1 29.1 595, 581, 598
E. coli DMSO - 20.7 2.5 - 21, 18, 23
Test item 33 21.7 3.5 1.0 25, 22, 18
100 26.7 3.2 1.3 28, 29, 23
333 25.0 3.5 1.2 23, 29, 23
1000 21.3 2.5 1.0 24, 21, 19
2500 16.0 2.0 0.8 18, 16, 14
5000 17.3 4.0 0.8 13, 21, 18
  4-NQO 5 1150.0 49.7 55.6 1202, 1145, 1103

1st experiment, standard plate incorporation assay with metabolic activation
Strain test group dose (mg/plate) mean revertants per plate standard deviation factor individual colony count
TA 1535 DMSO - 13.7 4.2 - 9, 17, 15
Test item 33 10.0 2.6 0.7 12, 11, 7
100 10.3 4.9 0.8 7, 8, 16
333 12.0 5.0 0.9 7, 17, 12
1000 9.3 3.1 0.7 6, 12, 10
2500 12.0 6.2 0.9 7, 10, 19
5000 10.3 4.0 0.8 8, 15, 8
  2-AA 2.5 271.3 11.2 19.9 274, 281, 259
TA 100 DMSO - 109.0 10.4 - 97, 115, 115
Test item 33 105.3 6.5 1.0 105, 99, 112
100 112.0 8.7 1.0 102, 117, 117
333 103.7 10.7 1.0 113, 92, 106
1000 94.7 9.7 0.9 103, 97, 84
2500 88.0 4.0 0.8 84, 88, 92
5000 101.0 18.1 0.9 103, 82, 118
  2-AA 2.5 2962.3 124.5 27.2 3086, 2964, 2837
TA 1537 DMSO - 9.7 2.1 - 8, 12, 9
Test item 33 12.3 2.5 1.3 12, 10, 15
100 11.0 1.0 1.1 12, 10, 11
333 12.7 4.5 1.3 13, 8, 17
1000 9.0 2.0 0.9 9, 11, 7
2500 6.3 2.3 0.7 5, 5, 9
5000 4.7 3.1 0.5 4, 8, 2
  2-AA 2.5 219.0 23.6 22.7 229, 236, 192
TA 98 DMSO - 24.0 4.4 - 26, 27, 19
Test item 33 23.7 6.7 1.0 31, 18, 22
100 21.7 4.2 0.9 23, 25, 17
333 22.0 11.3 0.9 35, 16, 15
1000 25.3 12.3 1.1 22, 15, 39
2500 19.0 4.6 0.8 14, 20, 23
5000 23.7 3.1 1.0 27, 21, 23
  2-AA 2.5 2681.7 189.9 111.7 2590, 2555, 2900
E. coli DMSO - 34.3 4.9 - 32, 31, 40
Test item 33 33.0 13.9 1.0 24, 26, 49
100 22.3 1.2 0.7 23, 23, 21
333 25.7 1.5 0.7 26, 27, 24
1000 24.0 7.2 0.7 30, 16, 26
2500 18.3 3.5 0.5 22, 15, 18
5000 16.3 7.8 0.5 10, 25, 14
  2-AA 60 90.3 13.3 2.6 75, 99, 97

2st experiment, preincorporation assay without metabolic activation
Strain test group dose (mg/plate) mean revertants per plate standard deviation factor individual colony count
TA 1535 DMSO - 10.3 3.1 - 11, 7, 13
Test item 33 11.7 1.5 1.1 12, 10, 13
100 11.0 4.6 1.1 7, 10, 16
333 7.7 0.6 0.7 8, 7, 8
1000 9.7 0.6 0.9 10, 9, 10
2500 11.3 3.5 1.1 15, 8, 11
5000 6.0 5.3 0.6 2, 12, 4
  MNNG 5.0 2405.0 253.9 232.7 2673, 2168, 2374
TA 100 DMSO - 99.3 16.2 - 89, 91, 118
Test item 33 103.7 10.0 1.0 100, 96, 115
100 89.7 8.4 0.9 80, 94, 95
333 92.3 6.4 0.9 97, 95, 85
1000 79.3 8.1 0.8 84, 84, 70
2500 94.0 6.6 0.9 101, 93, 88
5000 71.0 7.5 0.7 70, 79, 64
  MNNG 5.0 2405.3 260.1 24.2 2414, 2141, 2661
TA 1537 DMSO - 6.7 0.6 - 7, 7, 6
Test item 33 9.0 7.8 1.4 5, 4, 18
100 10.3 1.5 1.6 12, 9, 10
333 9.3 0.6 1.4 10, 9, 9
1000 11.3 5.7 1.7 16, 13, 5
2500 7.7 4.0 1.2 10, 3, 10
5000 5.0 2.0 0.8 3, 5, 7
  AAC 100 135.7 40.6 20.4 106, 182, 119
TA 98 DMSO - 15.0 1.0 - 14, 16, 15
Test item 33 17.7 4.6 1.2 15, 23, 15
100 20.0 7.9 1.3 17, 14, 29
333 14.7 1.2 1.0 14, 14, 16
1000 16.3 5.7 1.1 18, 10, 21
2500 15.3 8.3 1.0 22, 6, 18
5000 9.0 1.7 0.6 8, 8, 11
  NOPD 10 537.3 23.0 35.8 514, 560, 538
E. coli DMSO - 26.7 8.1 - 21, 36, 23
Test item 33 25.0 2.0 0.9 23, 27, 25
100 27.7 4.2 1.0 23, 31, 29
333 28.0 4.6 1.1 32, 23, 29
1000 18.0 1.7 0.7 17, 17, 20
2500 16.0 5.2 0.6 13, 13, 22
5000 14.0 3.0 0.5 11, 17, 14
  4-NQO 5 717.0 107.4 26.9 841, 656, 654

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 according to OECD TG 471 and GLP.

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

DOSE RANGE: 33 μg - 5000 μg/plate (SPT) 33 μg - 5000 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 weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 2500 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.

CONCLUSION: 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.