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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:
31 Oct - 30 Nov 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
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
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/bacht: Tank 25 20.08.2012

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature; temperature below 40 °C, light exclusion, protected against humidity
- Stability under test conditions: The stability of the test substance under storage conditions throughout the study period was guaranteed by the sponsor until 31 Jul 2013; the sponsor holds this responsibility

Method

Target gene:
his operon for S. typhimurium strains
trp operon for the E. coli strain
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:
other: TA 98: rfa-, uvrB-, R-factor; TA 100: rfa-, uvrB-, R-factor; TA 1535: rfa-, uvrB-; TA 1537: rfa-, uvrB; WP2: trp-; 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
Third experiment (preincubation test with and without metabolic activation, 3 plates/dose or control): 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate

Remark: due to contamination affecting the whole second experimental series, a third experiment was added.
Vehicle / solvent:
- Vehicle/solvent used: ultrapure water
- Justification for choice of solvent/vehicle: good solubility of the test item in the vehicle
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
Remarks:
ultrapure water
True negative controls:
no
Positive controls:
yes
Remarks:
with S9-mix
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
2.5 µg/plate in DMSO for TA 1535, TA 1537, TA 100, TA 98; 60 µg/plate in DMSO for E. coli WP2 uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
without S9-mix
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
Remarks:
5 µg/plate in DMSO for TA 1535 and TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
without S9-mix
Positive control substance:
9-aminoacridine
Remarks:
100 µg/plate in DMSO for TA 1537 Migrated to IUCLID6: (AAC)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
without S9-mix
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
5 µg/plate in DMSO for E. coli WP2 uvrA Migrated to IUCLID6: (4-NQO)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
(without S9-mix)
Positive control substance:
other: 4-nitro-o-phenylendiamine (NOPD)
Remarks:
10 µg/plate in DMSO for TA 98
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. As amino acid solution for the soft agar was used 0.5 mM histidine and 0.5 mM biotin for TA strains and 0.5 mM tryptophan for the E. coli strain.
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 Vogel-Bonner (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.
For the E. coli strain, plate test differed again in mixture of amino acid solution of the soft agar, the histidine component used for the TA strains being replaced by tryptophan.
Evaluation criteria:
An assay is accepted when the following criteria are met:
1.) number of colonies in the negative control is in the historical control range
2.) no indication of bacterial contamination (checked by sterility control)
3.) number of colonies in the positive controls are in the range of historical control data
4.) titer of viable bacteria is ≥ 10 E+8/mL

Toxicity is detected by:
1.) decrease in the number of revertants
2.) titer reduction
3.) clearing or diminution of the background lawn

Precipitation:
As long as no interference between precipitation and colony counting occurs is 5 mg/plate set as maximum dose even for relatively insoluble compounds.

A test chemical is to be considered as mutagenic when:
1.) increase of number of revertant colonies is reproducible and dose-related.
2.) in at least 1 tester strain doubling of colony counts with or without S-9 mix or after adding a metabolizing system is seen.

A test chemical is to be considered as non-mutagenic when:
1.) the number of revertants is inside the range of historical negative control data in 2 experiments performed independently from each other.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
occasionally observed depending on strain and test conditions from about 1000 μg/plate onward
Vehicle controls validity:
valid
Untreated negative controls validity:
other: sterility control, yes
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
occasionally observed depending on strain and test conditions from about 1000 μg/plate onward
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Remarks:
sterility control, yes
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
occasionally observed depending on strain and test conditions from about 1000 μg/plate onward
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Remarks:
sterility control, yes
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
occasionally observed depending on strain and test conditions from about 1000 μg/plate onward
Vehicle controls validity:
valid
Untreated negative controls validity:
other: sterility control, yes
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
occasionally observed depending on strain and test conditions from about 1000 μg/plate onward
Vehicle controls validity:
valid
Untreated negative controls validity:
other: sterility control, yes
Positive controls validity:
valid
Additional information on results:
No precipitation was detected.
No cytotoxic effects were seen in the standard plate test (SPT).
The first preincubation test (PIT) with and without metabolic activation was removed due to contamination and needed to be repeated. In the second PIT, a slight decrease in the number of his+ revertants indicating cytotoxicity could occasionally be seen depending on the strain and test conditions from about 1000 μg/plate onward.
Negative and positive controls were as expected and confirmed the validity and sensitivity of the test method and system.

Any other information on results incl. tables

Experiment 1: Standard plate-incorporation test

SPT without S9-Mix
 [mean no. of mutations/ plate]
Dosage [µg/plate] TA 1535 TA 100 TA 1537 TA 98 WP2 uvrA
Solvent control 11 54 7 24 88
33 11 47 10 23 86
100 12 53 7 22 102
333 11 53 7 26 93
1000 11 62 9 21 91
2500 11 55 6 21 101
5000 11 48 8 22 96
Respective positive control 1096 925 365 642 1102
SPT with S9-Mix
 [mean no. of mutations/ plate]
Dosage [µg/ plate] TA 1535 TA 100 TA 1537 TA 98 WP2 uvrA
Solvent control 11 68 12 29 96
33 12 62 13 29 100
100 13 58 14 29 109
333 12 64 10 34 100
1000 11 59 11 33 100
2500 12 59 13 27 100
5000 13 64 12 34 105
Respective positive control 210 1141 340 1190 417
Experiment 3: Preincubation test* PIT without S9-Mix
 [mean no. of mutations/ plate]
Dosage [µg/ plate] TA 1535 TA 100 TA 1537 TA 98 WP2 uvrA
Solvent control 17 48 9 20 109
33 16 45 8 20 109
100 16 50 7 22 105
333 17 48 8 16 105
1000 17 50 7 20 102
2500 10 50 6 19 103
5000 12 51 4 24 107
Respective positive control 1445 958 349 732 1056
PIT with S9-Mix
 [mean no. of mutations/ plate]
Dosage [µg/ plate] TA 1535 TA 100 TA 1537 TA 98 WP2 uvrA
Solvent control 12 57 8 22 118
33 11 53 7 21 118
100 14 60 8 25 121
333 12 57 9 24 116
1000 14 58 8 21 107
2500 11 55 9 22 100
5000 13 59 8 20 96
Respective positive control 446 1188 636 748 252
*Exp 2 not evaluated because of contamination

Applicant's summary and conclusion