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:
2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Physical state: liquid
- Storage condition of test material: Approximately 4ºC in the dark under nitrogen

Method

Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9
Test concentrations with justification for top dose:
Preliminary test (toxicity test): 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate.
Range-finding test: 50, 150, 5000, 1500 and 5000 ug/plate
Main test: 50, 150, 5000, 1500 and 5000 ug/plate

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: diemthyl sulphoxide
- Justification for choice of solvent/vehicle: The test material was immiscible in sterile distilled water at 50 mg/ml (the most concentrated stock solution) but was fully miscible in dimethyl sulphoxide.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Exposure duration: All of the plates were incubated at 37ºC for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria.

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the preliminary test, the test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Range finding test

Range-finding test with and without S9
  Number of revertants (mean number of colonies per plate)
S9 Mix Test substance concentration (ug/plate) TA100 Mean TA1535 Mean WP2uvrA- Mean TA98 Mean TA1537 Mean
- 0 74 89 22 27 24 21 24 20 11 10
90 32 16 17 7
102 26 22 19 11
- 50 95 97 29 32 17 17 16 17 8 8
90 37 19 16 7
105 30 15 19 9
- 150 94 95 21 24 12 14 15 23 8 10
C 25 13 15 8
95 15 17 38 15
- 500 118 105 24 29 20 23 25 23 7 9
87 34 21 22 15
110 30 29 22 6
- 1500 89 95 28 26 20 21 18 17 4 7
106 26 24 12 12
89 24 18 21 6
- 5000 97 106 33 34 20 20 19 12 5 6
111 30 18 7 10
109 39 22 11 4
                       
+ 0 85 89 13 21 21 21 24 24 16 11
93 11 25 25 10
89 10 16 23 6
+ 50 88 81 12 24 20 24 29 24 6 12
74 12 23 30 13
82 9 29 12 17
+ 150 85 91 11 15 15 15 25 26 15 20
89 6 17 30 19
98 7 13 23 26
+ 500 72 78 10 21 21 21 22 24 10 14
79 10 21 23 15
83 3 20 26 18
+ 1500 94 79 1 21 21 21 16 19 19 14
76 13 22 16 11
67 10 20 24 13
+ 5000 78 79 7 23 22 23 24 15 15 15
77 11 27 9 17
81 7 20 13 13
Positive controls ENNG ENNG ENNG 4NQO 9AA
Concentration (ug/plate) 3 5 2 0.2 80
without S9 436 469 193 191 828 858 120 128 1034 869
487 202 894 146 1160
484 179 851 118 412
Positive controls 2AA 2AA 2AA BP 2AA
Concentration (ug/plate) 1 2 10 5 2
With S9 767 834 187 221 314 282 166 214 222 250
845 245 277 223 273
891 232 252 254 254

Table 2. Main test

Main test with and without S9
  Number of revertants (mean number of colonies per plate)
S9 Mix Test substance concentration (ug/plate) TA100 Mean TA1535 Mean WP2uvrA- Mean TA98 Mean TA1537 Mean
- 0 84 94 28 23 20 21 17 18 10 9
88 17 18 18 9
111 25 24 19 9
- 50 94 93 29 22 20 22 21 20 8 12
86 22 20 16 17
98 29 26 24 10
- 150 109 107 30 21 17 21 17 14 6 8
116 32 24 15 8
97 29 33 9 9
- 500 112 109 32 18 16 18 19 20 10 8
103 30 20 21 4
113 22 19 21 10
- 1500 96 98 30 18 11 18 18 20 11 11
102 39 17 30 15
96 19 25 11 8
- 5000 113 110 31 13 5 13 6 11 5 6
100 28 23 8 6
118 32 11 18 6
                       
+ 0 103 98 10 10 36 30 21 27 15 19
96 10 26 29 20
96 11 27 31 21
+ 50 104 103 10 13 24 27 23 24 13 18
101 19 22 26 24
105 11 35 24 16
+ 150 106 104 11 9 19 21 27 25 11 14
105 9 24 18 19
100 7 19 29 13
+ 500 101 97 15 13 26 21 23 22 12 13
102 9 24 22 13
89 16 12 22 13
+ 1500 82 93 12 13 19 23 28 23 11 18
115 17 24 21 27
83 11 26 19 15
+ 5000 104 93 9 14 25 25 20 20 16 16
101 18 25 21 12
73 C 26 19 19
                       
Positive controls ENNG ENNG ENNG 4NQO 9AA
Concentration (ug/plate) 3 5 2 0.2 80
without S9 493 493 307 302 600 600 161 153 1145 1179
497 323 613 133 1131
488 277 588 165 1262
Positive controls 2AA 2AA 2AA BP 2AA
Concentration (ug/plate) 1 2 10 5 2
With S9 482 706 266 249 310 299 264 249 119 189
807 263 309 237 117
828 218 278 245 332

BP - Benzo(a)pyrene

2AA - 2-Aminoanthracene

ENNG - N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO - 4 -Nitroquinoline-1-oxide

9AA - 9 -Aminoacridine

C - Contaminated

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of this study the test material was considered to be non-mutagenic in the presence and absence of S9 activation.
Executive summary:

The study was performed to the requirements of OECD Guideline 471 under GLP conditions, to evaluate the potential mutagenicity of the test substance in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix.

A preliminary test was performed to determine the toxicity of the test material.A range-finding study was performed to to determine the doses used for the main test. In the main test, the plate incorporation method was used and was evaluated at a concentration of up to 5000 µg/plate. Positive controls appropriate for each strain, in the presence and absence of S9 -mix, were included. The test substance did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the strains tested, either in the presence or absence of S9-mix. The sensitivity of the test system and the metabolic activity of the S9-mix were clearly demonstrated by the increases in the numbers of revertant colonies induced by the positive control substances. It was concluded that, under the conditions of this assay, the test substance gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.