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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:
2015-06-19 to 2015-06-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Guideline and GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

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
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Salmonella typhimurium: histidine locus
Escherichia coli: tryptophan locus
Species / strain
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:
Phenobarbital/β-naphthoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
(NaN3), 10 μg/plate, without metabolic activation TA 1535, TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
(4-NOPD), without metabolic activation, 10 μg/plate in strain TA 98, 50 μg/plate in strain TA 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
(MMS), 2.0 μL/plate, without metabolic activation, WP2 uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
(2-AA), with metabolic activation, TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn
Evaluation criteria:
A test item was 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 was observed.
A dose dependent increase was considered biologically relevant if the threshold was exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold was 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 was not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data was not mandatory. Therefore no analysis was performed.

Results and discussion

Test results
Species / strain:
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:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate in the presence of S9 mix

ADDITIONAL INFORMATION ON CYTOTOXICITY:
experiment I: without S9: TA1537 (5000 μg/plate), TA100 (2500-5000 μg/plate), WP2uvrA (5000 μg/plate); with S9 mix: TA1535 and TA100 (1000-5000 μg/plate), TA1537 and TA98 and WP2uvrA (2500-5000 μg/plate)
experiment II: seen for all strains with and without S9 mix: 1000-5000 μg/plate
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Plate incorporation test (experiment 1):

Dose (µg/plate) Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
  TA1535 TA1537 TA98 TA100 WP2 uvrA
   
  Results without S9
DMSO 9 ± 1 9 ± 4 19 ± 6 147 ± 8 46 ± 8
Untreated 8 ± 2 9 ± 4 25 ± 2 152 ± 17 53 ± 1
3 10 ± 3 9 ± 2 21 ± 7 166 ± 21 49 ± 9
10 9 ± 4 8 ± 2 24 ± 7 154 ± 7 42 ± 6
33 14 ± 2 7 ± 2 25 ± 4 151 ± 17 47 ± 4
100 8 ± 2 7 ± 1 26 ± 5 150 ± 19 47 ± 3
333 13 ± 4 5 ± 0 23 ± 5 90 ± 19 43 ± 6
1000 15 ± 6 7 ± 1 17 ± 5 67 ± 3 33 ± 5
2500 11 ± 3 7 ± 1 17 ± 6 58 ± 1 23 ± 2
5000 11 ± 5 2 ± 2 14 ± 2 63 ± 10 16 ± 3
NaN3 (10) 1205 ± 87     2027 ± 45  
4-NOPD (10)     327 ± 17    
4-NOPD (50)   72 ± 12      
MMS (2.0 µL)         985 ± 46
   
  Results with S9
DMSO 13 ± 1 8 ± 0 27 ± 5 135 ± 1 58 ± 8
Untreated 13 ± 4 13 ± 3 33 ± 2 149 ± 4 63 ± 12
3 11 ± 1 9 ± 4 30 ± 5 144 ± 30 56 ± 5
10 11 ± 3 11 ± 3 27 ± 7 134 ± 16 60 ± 9
33 15 ± 2 12 ± 2 35 ± 3 150 ± 20 56 ± 6
100 12 ± 0 13 ± 1 27 ± 6 150 ± 28 66 ± 14
333 8 ± 3 15 ± 2 35 ± 2 110 ± 5 61 ± 17
1000 6 ± 2 4 ± 2 23 ± 1 57 ± 13 29 ± 9
2500 6 ± 2 1 ± 1 4 ± 1 19 ± 3 9 ± 3
5000 1 ± 1 0 ± 0 0 ± 0 0 ± 1 1 ± 1
2-AA (2.5) 446 ± 22 120 ± 9 3437 ± 519 3362 ± 163  
2-AA (10.0)         423 ± 41

Pre-incubation test (experiment 2:

Dose (µg/plate) Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
  TA1535 TA1537 TA98 TA100 WP2 uvrA
   
  Results without S9
DMSO 15 ± 1 10 ± 4 20 ± 4 118 ± 7 43 ± 6
Untreated 11 ± 4 10 ± 2 21 ± 4 148 ± 20 37 ± 1
3 8 ± 3 8 ± 2 21 ± 2 131 ± 10 37 ± 4
10 7 ± 1 8 ± 2 27 ± 1 129 ± 9 35 ± 8
33 11 ± 2 8 ± 4 18 ± 2 124 ± 12 41 ± 9
100 9 ± 2 9 ± 2 24 ± 3 106 ± 29 48 ± 8
333 9 ± 4 7 ± 2 21 ± 5 56 ± 5 38 ± 6
1000 4 ± 2 1 ± 1 0 ± 1 26 ± 23 13 ± 7
2500 2 ± 2 0 ± 0 0 ± 0 11 ± 11 11 ± 8
5000 0 ± 0 0 ± 0 0 ± 0 5 ± 8 0 ± 0
NaN3 (10) 1201 ± 21     2191 ± 176  
4-NOPD (10)     338 ± 16    
4-NOPD (50)   92 ± 4      
MMS (2.0 µL)         608 ± 17
   
  Results with S9
DMSO 11 ± 2 13 ± 1 37 ± 7 105 ± 14 48 ± 3
Untreated 9 ± 3 9 ± 1 39 ± 9 146 ± 8 42 ± 9
3 8 ± 1 11 ± 2 32 ± 3 101 ± 11 43 ± 8
10 7 ± 2 12 ± 5 33 ± 1 114 ± 5 51 ± 5
33 10 ± 4 15 ± 5 32 ± 3 108 ± 10 57 ± 6
100 13 ± 1 17 ± 3 36 ± 2 108 ± 5 54 ± 14
333 9 ± 4 13 ± 3 33 ± 4 63 ± 8 48 ± 1
1000 0 ± 1 1 ± 1 1 ± 1 2 ± 3 21 ± 5
2500 0 ± 1 0 ± 0 0 ± 0 0 ± 0 0 ± 1
5000 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0
2-AA (2.5) 420 ± 26 134 ± 18 4232 ± 148 3123 ± 114  
2-AA (10.0)         410 ± 17

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

An in vitro reverse mutation assay study (Ames) was conducted in Salmonella typhimurium and Escherichia coli strains. It was concluded that the test substance did not induce gene mutations and was considered to be non-mutagenic.
Executive summary:

An in vitro reverse mutation assay study (Ames) was conducted according to OECD Guideline 471 in Salmonella typhimurium and Escherichia coli strains. Experiment I was performed as a plate incorporation assay. Since a negative result was obtained in this experiment, experiment II was performed as a pre-incubation assay. All tests were conducted in triplicate and concentrations between 3 μg/plate and 5000 μg/plate were tested. Strains were exposed to the test substance with and without metabolic activation with rat liver S9 mix. Cytotoxicity was observed in the plate incorporation test without S9 in three strains (TA1537 (5000 μg/plate), TA100 (2500-5000 μg/plate), WP2uvrA (5000 μg/plate)) and with S9 mix in all strains (TA1535 and TA100 (1000-5000 μg/plate), TA1537 and TA98 and WP2uvrA (2500-5000 μg/plate)). In the pre-incubation test cytotoxicity was observed in all test strains independent of metabolic activation in the range from 1000 to 5000 μg/plate. No substantial increase in revertant colony numbers of any of the five tester strains was detected following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Therefore it was concluded that the test substance did not induce gene mutations and it was considered to be non-mutagenic.