Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 April - 21 Aug 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted: 21 Jul 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May, 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his operon (for S. typhimurium strains)
trp operon (for E. coli strain)
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
Test concentrations with justification for top dose:
Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment Ib (TA98): 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment 2: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation
Vehicle:
- Solvent/vehicle used: DMSO
- Justification for choice of solvent/ vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controls
Negative controls:
yes
Remarks:
untreated
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA)
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (Experiment 1 and 1b); preincubation (Experiment 2)

DURATION
- Preincubation period: 1 h
- Exposure duration: at least 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 (3 for TA 98) independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
Mean values and standard deviations were calculated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
see "remarks on result"
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Cytotoxicity:
Remarks:
Exp 1: at 333 µg/plate and above for TA 1537 and TA 100 with and without metabolic activation, at 1000 µg/plate and above for TA 98 with metabolic activation; Exp 1b: at 1000 µg/plate for TA 98 without metabolic activation; Exp 2: at 333 µg/plate and above for TA 1537, TA 98 and TA 100 with and without metabolic activation, at 1000 µg/plate and above for TA 1535 without metabolic activation
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate with S9 mix in experiment 1 and from 1000 to 5000 μg/plate with S9 mix in experiment 2 in all strains used. In experiment 1b no precipitation in the overlay agar in the test tubes was observed. No precipitation of the test item occurred in the overlay agar on the incubated agar plates in all experiments.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment is reported as Experiment I (all strains tested). In strain TA98 in the pre-experiment no colonies were observed at 333 μg/plate and above and the decrease was steep and unexpected. Therefore, this part of the experiment was repeated (Exp 1b). Based on the outcome of experiment 1b the results of strain TA 98 without S9 mix in the pre-experiment 1 were judged to be invalid due to a technical error. Therefore this part of the experiment is not reported.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxic effects, indicated by a reduced bacterial background lawn or decreased number of revertants (induction factor below 0.5), were observed in all strains used except srains TA 1535 and and WP2 uvrA in experiment I and II with and without S9 mix and in strain TA1537 in experiment I with and without metabolic activation. In Experiment 1 and 1b the strains TA1537, TA98 and TA100 showed reduced background growth. In Experiment 2 additionally strain TA1535 showed these quantitative signs of toxicity.

Any other information on results incl. tables

Table 2. Results experiment 1

Metabolic Activation Test Group Dose Level (per plate) Revertant Colony Counts (Mean ± SD)
      TA 1535 TA 1537 TA 98 TA 100 WP2 uvrA
Without Activation DMSO   12 ± 3 11 ± 4

1)

184 ± 13 43 ± 5
Untreated   9 ± 3 9 ± 5   200 ± 16 39 ± 7
  Test substance 3 µg 10 ± 5 14 ± 6   201 ± 30 37 ± 10
    10 µg 9 ± 3 9 ± 3   200 ± 8 38 ± 6
    33 µg 11 ± 3 10 ± 3   198 ± 8 37 ± 5
    100 µg 8 ± 2 8 ± 2   148 ± 11 40 ± 6
    333 µg 8 ± 3 14 ± 2R   38 ± 3R 44 ± 6
    1000 µg 11 ± 1 13 ± 2R   29 ± 5M R 36 ± 1
    2500 µg 10 ± 1 18 ± 6R   22 ± 8M R 48 ± 8
    5000 µg 9 ± 2 22 ± 6R   11 ± 1R M 38 ± 8
  NaN3 10 µg 1365 ± 40     2341 ± 189  
  4-NOPD 10 µg          
  4-NOPD 50 µg   70 ± 16      
  MMS 2.0 µL         972 ± 44
With Activation DMSO   11 ± 2 13 ± 5 32 ± 5 166 ± 11 47 ± 6
Untreated   11 ± 4 16 ± 6 44 ± 7 181 ± 17 58 ± 2
  Test substance 3 µg 12 ± 0 13 ± 1 35 ± 14 152 ± 23 50 ± 8
    10 µg 9 ± 2 12 ± 5 37 ± 10 161 ± 16 54 ± 3
    33 µg 15 ± 1 13 ± 2 28 ± 3 163 ± 10 64 ± 5
    100 µg 10 ± 4 11 ± 2 33 ± 6 173 ± 17 61 ± 12
    333 µg 11 ± 2 11 ± 2R 26 ± 3 52 ± 10R 45 ± 3
    1000 µg 11 ± 2 19 ± 4R 26 ± 5R 31 ± 5R 44 ± 6
    2500 µg 9 ± 3 19 ± 4R 13 ± 2M R 30 ± 6M R 37 ± 3
    5000 µg 8 ± 1 22 ± 3R 9 ± 4M R 21 ± 6M R 41 ± 5
  2-AA 2.5 µg 491 ± 21 204 ± 47 4024 ± 726 4041 ± 138  
  2-AA 10.0 µg         342 ± 13

1) experiment not valid; no results reported

NaN3: sodium azide

2AA: 2-Aminoanthracene

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

R: reduced background growth

M: manual count

Table 3: Results experiment 1b

Metabolic Activation Test Group Dose Level (per plate) Revertant Colony Counts (Mean  ±SD) 
      TA 98
Without Activation DMSO   24 ± 1
Untreated   19 ± 3
  Test substance 3 µg 27 ± 2
    10 µg 20 ± 3
    33 µg 25 ± 7
    100 µg 20 ± 5
    333 µg 22 ± 6
    1000 µg 18 ± 4R
    2500 µg 19 ± 2M R
    5000 µg 21 ± 6M R
  4-NOPD 10 µg 487 ± 16

4-NOPD: 4-nitro-o-phenylene-diamine

R: reduced background growth

M: manual count

Table 4. Results experiment 2

Metabolic Activation Test Group Dose Level (per plate) Revertant Colony Counts (Mean ±SD)
      TA 1535 TA 1537 TA 98 TA 100 WP2 uvrA
Without Activation DMSO   12 ± 2 10 ± 1 22 ± 7 139 ± 9 40 ± 7
Untreated   13 ± 3 8 ± 3 29 ± 7 161 ± 10 47 ± 5
  Test substance 3 µg     25 ± 3 131 ± 13  
    10 µg     25 ± 3 133 ± 3  
    33 µg 12 ± 3 7 ± 3 22 ± 6 134 ± 18 40 ± 3
    100 µg 8 ± 2 9 ± 2 15 ± 1 63 ± 17 38 ± 10
    333 µg 10 ± 3 8 ± 1R 21 ± 8R 31 ± 9R 37 ± 4
    1000 µg 9 ± 3R 3 ± 2R M 11 ± 1M R 14 ± 3M R 27 ± 5
    2000 µg   4 ± 1M R      
    2500 µg 10 ± 5R   6 ± 2M R 6 ± 1M R 40 ± 3
    3000 µg   2 ± 1M R      
    4000 µg   0 ± 1M R      
    5000 µg 11 ± 5R 0 ± 0M R 4 ± 2M R 0 ± 1M R 38 ± 6
  NaN3 10 µg 1334 ± 26     2333 ± 91  
  4-NOPD 10 µg     301 ± 18    
  4-NOPD 50 µg   99 ± 27      
  MMS 2.0 µL         603 ± 35
With Activation DMSO   13 ± 2 10 ± 2 38 ± 4 160 ± 12 53 ± 5
Untreated   12 ± 3 10 ± 3 41 ± 5 174 ± 26 56 ± 3
  Test substance 3 µg     32 ± 4 148 ± 3  
    10 µg     43 ± 3 161 ± 9  
    33 µg 12 ± 2 11 ± 4 33 ± 4 156 ± 6 48 ± 8
    100 µg 10 ± 2 10 ± 4 39 ± 2 106 ± 16 57 ± 7
    333 µg 11 ± 3 12 ± 5R 33 ± 8R 35 ± 5R 57 ± 15
    1000 µg 9 ± 3 10 ± 1M R 10 ± 2M R 10 ± 2M R 49 ± 6
    2000 µg   10 ± 3M R      
    2500 µg 8 ± 1   6 ± 2M R 12 ± 1M R 46 ± 5
    3000 µg   9 ± 2M R      
    4000 µg   7 ± 3M R      
    5000 µg 10 ± 6 5 ± 1M R 2 ± 0M R 2 ± 1M R 45 ± 4
  2-AA 2.5 µg 403 ± 86 141 ± 28 4973 ± 717 3765 ± 212  
  2-AA 10.0 µg         518 ± 29

NaN3: sodium azide

2AA: 2-Aminoanthracene

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

R: reduced background growth

M: manual count

Applicant's summary and conclusion

Conclusions:
Under the conditions of the conducted test the substance was not mutagenic in any of the five bacterial strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation.