Registration Dossier

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:
16 Feb 2012 - 02 Mar 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted according to GLP (2-AA was the only positive control used with metabolic activation).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
yes
Remarks:
2-AA was the only positive control used for metabolic activation
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
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): GSID 3056-1
- Physical state: solid, brownish
- Analytical purity: 98% (Analytical Report 11L00523)
- Lot/batch No.: 1470 VB04
- Storage condition of test material: room temperature
- Storage stability: The stability of the test substance under storage conditions throughout the study period was guaranteed until 06 Oct 2013 (as indicated by the sponsor, and 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, triplicate): 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Second experiment (preincubation test with and without metabolic activation, triplicate): 0, 10, 33, 100, 333, 1000 and 2500 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Due to the limited solubility of the test substance in ultrapure water, acetone was used as vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
(sterility control)
Negative solvent / vehicle controls:
yes
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
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
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)
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)
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:
In the standard plate test, tubes were filled with 2mL portions of soft agar and kept in a water bath at 42-45°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 with S. typhimurium strains were were poured onto Vogel-Bonner (minimal glucose agar plates) plate and incubated for 48 - 72 hrs in the dark at 37°C.
After mixing samples with the E.coli train were poured onto SA1 selective agar plates ( minimal agar prepared according to Green and Muriel, Mut Res 38: 3-32, 1976) and incubated for 48 - 72 hrs in the dark at 37°C.

For the preincubation test 0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL of the S9 mix were incubated at 37°C for 20 minutes (Yahagi et al., Mut Res 48: 121-129,1977; 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). 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.
Triplicate testing was done.
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+9/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
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No increase in number of revertants was observed.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Decrease in the number of his+ or trp+ revertants, slight reduction in the titer observed depending on the strain and test conditions from about 2 500 μg/plate onward.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No increase in number of revertants was observed.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, slight reduction in the titer, observed depending on the strain and test conditions from about 1 000 μg/plate onward.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Precipitation:
Precipitation of the test substance was observed in the standard plate test from 333 μg/plate onward (without S9 mix) and from 1000 μg/plate onward (with S9 mix).
Precipitation of the test substance was observed in the preincubation test from 1000 μg/plate onward with and without S9 mix.

Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table1: Results of Experiment I, Standard Plate Test

 With or  Test substance  Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)            
 without  concentration  Base-pair substitution type        Frameshift type   
 S9 mix  (µg/plate)  TA100  TA1535  E.coli WP2  TA98  TA1537
 -  acetone  68 ± 7  12 ± 2  44 ± 3  19 ± 3  6 ± 1
 -  33  78 ± 8  11 ± 2  43 ± 3  20 ± 3  6 ± 1
 -  100  79 ± 6  12 ± 1  42 ± 11  17 ± 2  7 ± 2
 -  333  75 ± 10  12 ± 2  41 ± 4  19 ± 2  6 ± 1
 -  1000  69 ± 6  11 ± 2  40 ± 7  17 ± 2  5 ± 2
 -  2500  40 ± 12  7 ± 1  28 ± 2  12 ± 4  3 ± 2
 -  5000  6 ± 1  4 ± 2  21 ± 6  3 ± 1  1 ± 0
 positive controls, -S9  Name  MNNG  MNNG  4 -NQO  NOPD  AAC
   Concentration [µg/plate]  5.0 5.0  5.0  10  2.5
   Mean No. of colonies/plate (average of 3 ± SD)  672 ± 93  637 ± 23  650 ± 44  846 ± 79  410 ± 20
     TA100  TA1535  E.coli WP2  TA98  TA1537
 +  acetone  79 ± 7  13 ± 1  48 ± 3  22 ± 3  7 ± 1
 +  33  81 ± 7  13 ± 3  44 ± 10  28 ± 4  7 ± 1
 +  100  77 ± 2  12 ± 1  48 ± 2  22 ± 2  9 ± 0
 +  333  78 ± 10  13 ± 2  47 ± 7  25 ± 4  6 ± 1
 +  1000  81 ± 4  12 ± 2  45 ± 6  21 ± 5  7 ± 1
 +  2500  58 ± 21  5 ± 2  35 ± 7  16 ± 2  3 ± 1
 +  5000  15 ± 7  2 ± 2  29 ± 3  7 ± 2  1 ± 1
 positive controls, +S9  Name  2 -AA  2 -AA  2 -AA  2 -AA  2 -AA
   Concentration [µg/plate]  2.5  2.5  60  2.5  100
   Mean No. of colonies/plate (average ± SD)  757 ± 43  126 ± 18  261 ± 28  651 ± 63  105 ± 5

Table2: Results of Experiment II, Preincubation Test

 With or  Test substance  Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)            
 without  concentration  Base-pair substitution type        Frameshift type   
 S9 mix  (µg/plate)  TA100  TA1535  E.coli WP2  TA98  TA1537
 -  acetone  74 ± 6  12 ± 2  44 ± 4  18 ± 3  7 ± 1
 -  10  79 ± 8  11 ± 2  42 ± 8  18 ± 4  7 ± 2
 -  33  72 ± 1  12 ± 4  38 ± 5  18 ± 7  8 ± 2
 -  100  73 ± 3  12 ± 1  42 ± 6  17 ± 3  6 ± 2
 -  333  72 ± 10  13 ± 2  43 ± 6  16 ± 2  7 ± 1
 -  1000  61 ± 11  9 ± 2  34 ± 6  11 ± 3  5 ± 3
 -  2500  B/P  B/P  13 ± 3  B/P  B/P
 positive controls, -S9  Name  MNNG  MNNG  4 -NQO  NOPD  AAC
   Concentration [µg/plate]  5.0 5.0  5.0  10  100
   Mean No. of colonies/plate (average of 3 ± SD)  672 ± 93  721 ± 48  591 ± 39  566 ± 33  359 ± 34
     TA100  TA1535  E.coli WP2  TA98  TA1537
 +  acetone  82 ± 10  13 ± 3  44 ± 9  27 ± 6  9 ± 1
 +  10  88 ± 14  11 ± 1  44 ± 12  28 ± 8  9 ± 1
 +  33  88 ± 7  13 ± 4  47 ± 8  28 ± 12  8 ± 3
 +  100  81 ± 8  14 ± 2  44 ± 7  29 ± 7  9 ± 3
 +  333  82 ± 11  12 ± 3  50 ± 4  29 ± 6  8 ± 1
 +  1000  70 ± 6  13 ± 2  31 ± 9  25 ± 4  8 ± 4
 +  2500  52 ± 6  8 ± 2  18 ± 4  19 ± 3  6 ± 1
 positive controls, +S9  Name  2 -AA  2 -AA  2 -AA  2 -AA  2 -AA
   Concentration [µg/plate]  2.5  2.5  60  2.5  2.5
   Mean No. of colonies/plate (average ± SD)  860 ± 67  136 ± 19  258 ± 17  599 ± 47  120 ± 13

P = Precipitation; B = Reduced Background growth

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

CLP: not classified
DSD: not classified