Registration Dossier

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well documented and reported study fully adequate for assessment. The study was conducted according to internationally accepted technical guidelines and in compliance with GLP in a recognized contract research organization. Study according to OECD guideline 471 under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: white solid
- Analytical purity: >99%

Method

Target gene:
uvrB
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain characteristics:
other: essential amino acid requiring strains
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 mix from male Wistar rats treated with phenobarbital and beta-Naphthoflavone for enzyme induction.
Species / strain:
E. coli WP2 uvr A
Additional strain characteristics:
other: essential amino acid requiring strains
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 mix from male Wistar rats treated with phenobarbital and beta-Naphthoflavone for enzyme induction.
Test concentrations with justification for top dose:
Experiment 1 (Plate Incorporation Test): 33, 100, 333, 1000, 2500, 5000 µg/plate
Experiment 2 (Pre-Incubation Test): 33, 100, 333, 1000, 2500, 5000 µg/plate
Vehicle:
DMSO
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
Positive control substances for tests with metabolic activation (S9 mix)
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: sodium azide, 4.nitro-o-phenylene-diamine, methyl methane sulfonate
Remarks:
Positive control substances for tests without metabolic activation (S9 mix). All of them are well established reference mutagens.
Details on test system and conditions:
Standard Plate Incorporation Tests were performed in both experiments (Experiments 1 and 2) and both experiments were conducted without and with metabolic activation (S9 mix).

The following positive controls were used to check mutability of the bacteria and activity of the S9 mix:

Without metabolic activation (S9 mix):

Sodium azide, NaN3:
- 10 μg/plate, dissolved in water deionised: - strains: TA 1535, TA 100

4-nitro-o-phenylene-diamine, 4-NOPD::
- 10 μg/plate, dissolved in DMSO: - strain: TA 98
- 50 µg/plate, dissolved in DMSO: - strain: TA 1537

Mehyl methane sulfonate, MMS:
- 5 μg/plate, dissolved in water deionised: - strain: WP2 uvrA

With metabolic activation (S9 mix):

2-Aminoanthracene, 2-AA:
- 2.5 μg/plate, dissolved in DMSO: - strains: TA 1535, TA 1537, TA 98, TA 100
- 10 μg/plate, dissolved in DMSO: - strain: WP2 uvrA
Evaluation criteria:
The test chemical is considered to exhibit mutagenic activity in this assay if the following criteria are met:
A reproducible increase in revertant colony number, (i.e. at least twice for strains TA 100, TA 98 and WP2 uvrA and at least three times for strains TA 1535 and TA 1537 the concurrent vehicle controls), with some evidence of a positive dose-response relationship. Such positive response in at least one tester strain without or with metabolic activation (S9 mix.) is sufficient for concluding mutagenic activity.

A test substance is considered non-mutagenic in this test if:
Exposure to a test substance does not produce a reproducible increase in revertant colony numbers.
Statistics:
not required

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
in both experiments (Experiment 1 and 2)
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: in both experiments (Experiment 1 and 2)
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
in both experiments (Experiment 1 and 2)
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: in both experiments (Experiment 1 and 2)

Any other information on results incl. tables

EXPERIMENT 1: Plate Incorporation Test

Strain: TA 1535

Without S9 mix

 Concentration [µg/plate]    Plate      Revertants/plate  
   1  2  3  mean  s.d.  factor*
 Negative Ctrl  9  9  9  9  0.0  
 Solvent Ctrl  10  10  11  10  0.6  1.0
 Positive Ctrl  915  939  816  890  65.2  86.1
 33  10  8  9  9  1.0  0.9
 100  10  13  9  11  2.1  1.0
 333  11  9  10  10  1.0  1.0
 1000  5  6  8  6  1.5  0.6
 2500  5  8  9  7  2.1  0.7
 5000  13  15  10  13  2.5  1.2

With S9 mix

 Concentration [µg/plate]    Plate    Revertants/plate  
   1  2  3  mean  s.d.  factor*
 Negative Ctrl  12  10  11  11  1.0  
 Solvent Ctrl  16  16  9  14  4.0  1.0
 Positive Ctrl  60  61  130 84  40.1  6.1
 33  9  11  10  10  1.0  0.7
 100  14  10 11  12  2.1  0.9
 333  10 8  5  8  2.5  0.6
 1000  6 5 6  6  0.6  0.4
 2500  8 9  8  8  0.6  0.6
 5000  7  5  9  7  2.0  0.5

*: enhancement factor = (sum of revertants per conc. test article) / (sum of revertants per solvent ctrl)

Strain: TA 1537

Without S9 mix

 Concentration [µg/plate]    Plate    Revertants/plate  
   1  2  3  mean  s.d.  factor*
 Negative Ctrl  10 7  9  9  1.5  
 Solvent Ctrl  11 9  11  10  1.2  1.0
 Positive Ctrl  123  107 108  113  9.0  10.9
 33  9  15  19  14  5.0  1.4
 100  10  11  5  9  3.2  0.8
 333  14  7  4  8  5.1  0.8
 1000  10  14  12  12  2.0  1.2
 2500  9  7  12  9  2.5  0.9
 5000  8  8  11  9  1.7  0.9

With S9 mix

 Concentration [µg/plate]    Plate    Revertants/plate  
   1  2  3  mean  s.d.  factor*
 Negative Ctrl  14  9  10  11  2.6  
 Solvent Ctrl  11  11  17  13  3.5  1.0
 Positive Ctrl  77  95  70 81  12.9  6.2
 33  11 13 15  13  2.0  1.0
 100  13  12 11  12  1.0  0.9
 333  13 12  10  12  1.5  0.9
 1000  13 8 11  11  2.5  0.8
 2500  10 9  10  10  0.6  0.7
 5000  12  11  14  12  1.5  0.9

*: enhancement factor = (sum of revertants per conc. test article) / (sum of revertants per solvent ctrl)

Strain: TA 98

Without S9 mix

 Concentration [µg/plate]    Plate    Revertants/plate  
   1  2  3  mean  s.d.  factor*
 Negative Ctrl  19 18  15  17  2.1  
 Solvent Ctrl  16 14  15  15  1.0  1.0
 Positive Ctrl  199  206 223  209  12.3  14.0
 33  5  20  15  13  7.6  0.9
 100  17  14 23  18  4.6  1.2
 333  15  11  18  15  3.5  1.0
 1000  9  28  14  17  9.8  1.1
 2500  7  11  11  10  2.3  0.6
 5000  11  11  9  10  1.2  0.7

With S9 mix

 Concentration [µg/plate]    Plate    Revertants/plate  
   1  2  3  mean  s.d.  factor*
 Negative Ctrl  26  26  27  26  0.6  
 Solvent Ctrl  18  15  17  17  1.5  1.0
 Positive Ctrl  619  502  507 543  66.2  32.6
 33  22 19 12  18  5.1  1.1
 100  20  27 19  22  4.4  1.3
 333  32 19  36  29  8.9  1.7
 1000  29 32 19  27  6.8  1.6
 2500  22 25  32  26  5.1  1.6
 5000  24  15  20  20  4.5  1.2

*: enhancement factor = (sum of revertants per conc. test article) / (sum of revertants per solvent ctrl)

Strain: TA 100

Without S9 mix

 Concentration [µg/plate]    Plate    Revertants/plate  
   1  2  3  mean  s.d.  factor*
 Negative Ctrl  80 91  78  83  7.0  
 Solvent Ctrl  98 91  92  94  3.8  1.0
 Positive Ctrl  1216  1135 1117  1156  52.7  12.3
 33  80  75  89  81  7.1  0.9
 100  97  75 88  87  11.1  0.9
 333  96  86  100  94  7.2  1.0
 1000  109  104  109  107  2.9  1.1
 2500  85  106  98  96  10.6  1.0
 5000  101  102  112  105  6.1  1.1

With S9 mix

 Concentration [µg/plate]    Plate    Revertants/plate  
   1  2  3  mean  s.d.  factor*
 Negative Ctrl  77  88  93  86  8.2  
 Solvent Ctrl  89  87  81  86  4.2  1.0
 Positive Ctrl  457  570  476 501  60.5  5.8
 33  90 81 80  84  5.5  1.0
 100  85  97 88  90  6.2  1.1
 333  74 75  87  79  7.2  0.9
 1000  75 82 91  83  8.0  1.0
 2500  92 102 99  98  5.1  1.1
 5000  84  97  78  86  9.7  1.0

*: enhancement factor = (sum of revertants per conc. test article) / (sum of revertants per solvent ctrl)

Strain: WP2 uvrA

Without S9 mix

 Concentration [µg/plate]    Plate    Revertants/plate  
   1  2  3  mean  s.d.  factor*
 Negative Ctrl  40 28  27  32  7.2  
 Solvent Ctrl  29 29  35  31  3.5  1.0
 Positive Ctrl  795  561 821  726  143.2  23.4
 33  29  35  42  35  6.5  1.1
 100  21  29 36  29  7.5  0.9
 333  24  32  39  32  7.5  1.0
 1000  19  28  22  23  4.6  0.7
 2500  22  25  17  21  4.0  0.7
 5000  28  16  19  21  6.2  0.7

With S9 mix

 Concentration [µg/plate]    Plate    Revertants/plate  
   1  2  3  mean  s.d.  factor*
 Negative Ctrl  34  34  41  36  4.0  
 Solvent Ctrl  35  36  35  35  0.6  1.0
 Positive Ctrl  139  140  186 155  26.9  4.4
 33  31 30 32  31  1.0  0.9
 100  37  22 32  30  7.6  0.9
 333  31 31  38  33  4.0  0.9
 1000  29 24 33  27  4.9  0.8
 2500  25 26 17  23  4.9  0.6
 5000  19  24  25  23  3.2  0.6

*: enhancement factor = (sum of revertants per conc. test article) / (sum of revertants per solvent ctrl)

EXPERIMENT 2: Pre-Incubation Test

Strain: TA 1535

Without S9 mix

 Concentration [µg/plate]    Plate      Revertants/plate  
   1  2  3  mean  s.d.  factor*
 Negative Ctrl  9  14  14  12  2.9  
 Solvent Ctrl  19  15  18  17  2.1  1.0
 Positive Ctrl  607  629  608  615  12.4  35.5
 33  6  10  13  10  3.5  0.6
 100  11  14  18  14  3.5  0.8
 333  20  11  9  13  5.9  0.8
 1000  13  14  14  14  0.6  0.8
 2500  11  10  14  12  2.1  0.7
 5000  8  11  13  11  2.5  0.6

With S9 mix

 Concentration [µg/plate]    Plate    Revertants/plate  
   1  2  3  mean  s.d.  factor*
 Negative Ctrl  8  8  10  9  1.2  
 Solvent Ctrl  10  11  10  10  0.6  1.0
 Positive Ctrl  81  77  91 83  7.2  8.0
 33  7  15  10  11  4.0  1.0
 100  9  7 7  8  1.2  0.7
 333  8 7  10  8  1.5  0.8
 1000  12 15 9  12  3.0  1.2
 2500  3 8  12  8  4.5  0.7
 5000  8  8  6  7  1.2  0.7

*: enhancement factor = (sum of revertants per conc. test article) / (sum of revertants per solvent ctrl)

Strain: TA 1537

Without S9 mix

 Concentration [µg/plate]    Plate      Revertants/plate  
   1  2  3  mean  s.d.  factor*
 Negative Ctrl  5  6  11  7  3.2  
 Solvent Ctrl  5  7  12  8  3.6  1.0
 Positive Ctrl  80  77  78  78  1.5  9.8
 33  8  3  3  5  2.9  0.6
 100  11  4  12  9  4.4  1.1
 333  8  8  8  8  0.0  1.0
 1000  9  9  9  9  0.0  1.1
 2500  9  12  23  15  7.4  1.8
 5000  12  4  13  10  4.9  1.2

With S9 mix

 Concentration [µg/plate]    Plate    Revertants/plate  
   1  2  3  mean  s.d.  factor*
 Negative Ctrl  10  10  11  10  0.6  
 Solvent Ctrl  9  4  10  8  3.2  1.0
 Positive Ctrl  31  53  54 46  13.0  6.0
 33  15  11  8  11  3.5  1.5
 100  13  18 11  14  3.6 1.8
 333  14 11  10  12  2.1  1.5
 1000  10 11 11  11  0.6  1.4
 2500  15 10  8  11  3.6  1.4
 5000  9  7  8  8  1.0  1.0

*: enhancement factor = (sum of revertants per conc. test article) / (sum of revertants per solvent ctrl)

Strain: TA 98

Without S9 mix

 Concentration [µg/plate]    Plate      Revertants/plate  
   1  2  3  mean  s.d.  factor*
 Negative Ctrl  15  15  15  15  0.0  
 Solvent Ctrl  16  16  11  14  2.9  1.0
 Positive Ctrl  172  151  135  153  18.6  10.7
 33  13  17  8  13  4.5  0.9
 100  13  13  18  15  2.9  1.0
 333  15  12  15  14  1.7  1.0
 1000  12  12  11  12  0.6  0.8
 2500  7  8  8  8  0.6  0.5
 5000  11  3  17  10  7.0  0.7

With S9 mix

 Concentration [µg/plate]    Plate    Revertants/plate  
   1  2  3  mean  s.d.  factor*
 Negative Ctrl  14  27  27  23  7.5  
 Solvent Ctrl  12  25  19  19  6.5  1.0
 Positive Ctrl  184  134  143 154  26.7  8.2
 33  23  17  19  20  3.1  1.1
 100  17  18 24  20  3.8 1.1
 333  21 14  11  15  5.1  0.8
 1000  10 17 15  14  3.6  0.8
 2500  15 9  12  12  3.0  0.6
 5000  18  10  10  13  4.6  0.7

*: enhancement factor = (sum of revertants per conc. test article) / (sum of revertants per solvent ctrl)

Strain: TA 100

Without S9 mix

 Concentration [µg/plate]    Plate      Revertants/plate  
   1  2  3  mean  s.d.  factor*
 Negative Ctrl  72  75  81  76  4.6  
 Solvent Ctrl  67  106  98  90  20.6  1.0
 Positive Ctrl  379  372  396  382  12.3  4.2
 33  48  48  40  45  4.6  0.5
 100  31  43  15  30  14.0  0.3
 333  25  23  15  21  5.3  0.2
 1000  45  24  24  31  12.1  0.3
 2500  11  11  13  12  1.2  0.1
 5000  16  16  12  15  2.3  0.2

With S9 mix

 Concentration [µg/plate]    Plate    Revertants/plate  
   1  2  3  mean  s.d.  factor*
 Negative Ctrl  85  78  74  79  5.6  
 Solvent Ctrl  86  81  69  79  8.7  1.0
 Positive Ctrl  446  489  432 456  29.7  5.8
 33  19  11  12  14  4.4  0.2
 100  8  12 7  9  2.6 0.1
 333  14 13  13  13  0.6  0.2
 1000  29 2 9  13  14.0  0.2
 2500  19 6  9  11  6.8  0.1
 5000  9  8  17  11  4.9  0.1

*: enhancement factor = (sum of revertants per conc. test article) / (sum of revertants per solvent ctrl)

Strain: WP2 uvrA

Without S9 mix

 Concentration [µg/plate]    Plate      Revertants/plate  
   1  2  3  mean  s.d.  factor*
 Negative Ctrl  27  28  27  27  0.6  
 Solvent Ctrl  28  25  29  27  2.1  1.0
 Positive Ctrl  166  147  159  157  9.6  5.8
 33  20  22  21  21  1.0  0.8
 100  27  23  29  26  3.1  1.0
 333  27  22  19  22  4.0  0.8
 1000  22  17  9  16  6.6  0.6
 2500  25  24  18  22  3.8  0.8
 5000  18  19  23  20  2.6  0.7

With S9 mix

 Concentration [µg/plate]    Plate    Revertants/plate  
   1  2  3  mean  s.d.  factor*
 Negative Ctrl  33  27  30  30  3.0  
 Solvent Ctrl  34  44  21  33  11.5  1.0
 Positive Ctrl  149  159  155 154  5.0  4.7
 33  28 35  34  32  3.8  1.0
 100  27  27 32  29  2.9 0.9
 333  11 31  32  25  11.8  0.7
 1000  26 35 19  27  8.0  0.8
 2500  19 22  20  20  1.5  0.6
 5000  24  27  27  26  1.7  0.8

*: enhancement factor = (sum of revertants per conc. test article) / (sum of revertants per solvent ctrl)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
ambiguous without metabolic activation

The test item is considered as non-mutagenic in the bacterial reverse mutation assay. Based on these results, the test item does not have to be classified according to DSD (Directive 67/548/EEC) or CLP (Regulation (EC) No. 1272/2008).
Executive summary:

The test article was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation (S9 mix). Each concentration and the controls were tested in triplicate. The test article was tested at the following concentrations: 33, 100, 333, 1000, 2500, and 5000 µg/plate. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No relevant toxic effects, evident as a reduction in the number of revertants, occured in experiment I with and without metabolic activation. In experiment II, toxic effects were observed from 100 µg/plate up to 5000 µg/plate without S9 mix and from 33 µg/plat up to 5000 µg/plate wit S9 mix in strain TA 100.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test article 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.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.