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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:
2010-01-14 to 2010-03-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Benzene, mono C10-13-alkyl derivs, distn. residues, sulfonated
IUPAC Name:
Benzene, mono C10-13-alkyl derivs, distn. residues, sulfonated
Details on test material:
- Name of test material (as cited in study report): Benzene, mono C10-13-alkyl derivs, distn. resiudes, sulfonated
- Substance type: product
- Physical state: dark brown, viscous liquid
- Storage condition of test material: storage at room temperature

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 activated liver homogenate from male Sprague-Dawley rats
Test concentrations with justification for top dose:
- 1st experiment: 5, 1.581, 0.5, 0.158 and 0.05 mg/plate with and without metabolic activation
- 2nd experiment: 5, 2.5, 1.25, 0.63, 0.31 and 0.16 mg/plate with metabolic activation, with strain TA 1537 in addition 0.08 mg/plate with metabolic activation; resp. 1, 0.5, 0.25, 0.13 0.06 and 0.03 mg/plate without metabolic activation
- 3rd experiment: 0.86, 0.72, 0.60, 0.50, 0.42, 0.35 and 0.29 mg/plate without metabolic activation , only TA 1535
All concentrations were applied corresponding to the sulphonated material
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not given
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
sterile deionized water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 50 µg/mL 2-Nitrofluorne with TA 98; 10 µg/mL Sodiumazide with TA 100 and TA 1535; 1.5 µL/mL Cumene hydroperoxide (ca. 80%) with TA 102; 1.5 mg/mL 9-Aminoacridine with TA 1537
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
sterile deionized water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 100 µg/mL with all tester strains; additionally 0.5 mg/mL Benz-a-pyrene with TA 98
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 per concentration

DETERMINATION OF CYTOTOXICITY
- Method: growth of micro-colonies in the background in comparison to the negative control by estimating the diameter and number of the micro-colonies under the microscope

OTHER EXAMINATIONS:
- Counting of revertants: with a semi-automatic colony counter (Ernst Schütt, jun., Göttingen, Germany)
- Titre of bacteria in the overnight culture: determined by counting colonies formed upon plating an aliquot of a 10E6-fold dilution with NaCl-solution and confirming the concentration by correlating the turbidity of the culture measured photometrically

OTHER:
- Qualitiy assurance of the Salmonella strains: Every half year the bacteria were checked concerning their genotype (sensitivity to crystal violet, sensitivity to ultraviolet light, testing for ampicillin resistance).
Evaluation criteria:
Criteria for validity of the test:
- the titre of the bacterial culture is sufficient (about 10E9 bacteria/mL)
- the positive control reach at least a doubling of the revertants in comparison to the negative controls
- the numbers of revertants in the negative controls are within the historical ranges (according to Venitt et al. (1984) and laboratory specific historical min-max data)
Criteria for a positive result:
A sample is judged to be mutagenic
- if there is a concentration-related increase over the range tested
- if there is a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation
- if a statistically significant difference between the mean values can be demonstrated in the case of a reproducible increase of the number of revertants
- if at least a doubling of revertant colonies with the sample compared to the control can be observed (Induction rate (IR) >= 2).
Statistics:
Calculation of the mean value and standard deviation of the replicates (Microsoft Excel), t-test with Bonferri adjustment and Wilcoxon rang sum test.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
for details see tables below
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA100 and TA 102
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
exception: cytotoxicity observed at the highest concentration with TA 98 only in the first experiment
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the first experiment with the stain TA 1535 a maximum induction rate of 2.1 was obtained with 0.5 mg/plate. In the second experiment, which was performed independently, this result was confirmed. All other concentrations tested were inconspicuous. In both studies this increase was not statistically significant and the increase did not follow any dose-response curve. To confirm this ambiguous result a third test with a very narrow concentration range was performed. In this experiment no increase of revertant colonies at all could be observed.

In the experiment without metabolic acitvation the test item was toxic for the bacteria at higher concentrations. Strain TA 102 turned out to be most sensitive for cytotoxicity. Here up to 250 µg/plate the test item was toxic.

The numbers of the revertants in the negative controls were within the historical ranges.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Mean values of the number of revertants and the induction factors obtained in the 1st experiment 
Concentration S9 mix TA 98 TA 100 TA 102 TA 1535 TA 1537
    mean RSD
[%]
IR mean RSD
[%]
IR mean RSD
[%]
IR mean RSD
[%]
IR mean RSD
{%]
IR
Negative control - 20 26.5 - 77 8.3 - 206 12 - 15 19.9 - 7 24.2 -
Solvent control - 21 10.4 - 82 13.7 - 194 6.7 - 10 29.2 - 6 37 -
Positive control - 159 7.3 7.7 347 7.4 4.2 1071 4.6 5.5 180 31.1 18.0 289 2 105.2
5 mg/plate - 3 * 57.3 0.1 44 * 8.2 0.5 6 * 115.5 0 3 21.7 0.3 2 * 0 0.4
1.581 mg/plate - 11 * 20.4 0.5 64 0.9 0.8 67 * 39.6 0.3 9 34.4 0.9 3 0 0.5
0.5 mg/plate - 15 10 0.7 75 4.7 0.9 123 * 12 0.6 21 45.9 2.1 7 14.3 1.3
0.158 mg/plate - 23 2.5 1.1 76 6.2 0.9 134 (*) 11.9 0.7 11 27 1.1 6 27 1.0
0.05 mg/plate - 24 15 1.2 71 1.6 0.9 143 15.8 0.7 12 8.3 1.2 7 37.7 1.2
                                 
Negative control + 23 15.4 - 92 8.3 - 223 6.2 - 11 19.3 - 6 20.4 -
Solvent control + 21 27 - 84 13.4 - 188 16.3 - 13 19.9 - 7 39.8 -
Positive control (2-AA) + 605 16.6 28.3 864 0.7 10.3 427 2.6 2.3 177 27.2 14.0 589 2 81.9
Positive control (B-a-P) + 223 25.5 10.4                        
5 mg/plate + 17 (*) 15.6 0.8 54 12 0.6 235 5.6 1.3 8 32.8 0.6 2 65.5 0.3
1.581 mg/plate + 23 22.6 1.1 86 8.7 1.0 336 7.6 1.8 9 13.3 0.7 5 43.3 0.7
0.5 mg/plate + 23 17.3 1.1 79 10.6 0.9 338 7.5 1.8 11 47.2 0.8 6 48.2 0.9
0.158 mg/plate + 26 9.6 1.2 76 12.2 0.9 203 3.1 1.6 9 11.1 0.7 10 6 1.3
0.05 mg/plate + 27 22.1 1.3 83 8.5 1.0 248 7.4 1.3 12 33.8 1.0 11 21.7 1.5
RSD = relative standard deviation   IR = Induction Rate   * = cytotoxic effects (background growth)
Table 2: Mean values of the number of revertants and the induction factors obtained in the 2nd experiment 
Concentration S9 mix TA 98 TA 100 TA 102 TA 1535 TA 1537
    mean RSD
[%]
IR mean RSD
[%]
IR mean RSD
[%]
IR mean RSD
[%]
IR mean RSD
{%]
IR
Negative control - 25 20.4 - 102 14.3 - 261 4.2 - 13 37.2 - 7 24.2 -
Solvent control - 35 15.5 - 76 8.5 - 175 25.3 - 8 31.9 - 8 48 -
Positive control - 166 11.3 4.8 174 3.7 2.3 521 6.0 3.0 104 16.7 13.0 638 44.7 83.9
1 mg/plate - 10 (*) 15.8 0.3 46 (*) 32.3 0.6 49 (*) 47.6 0.3 9 75.1 1.1 5 24.7 0.6
0.5 mg/plate - 13 (*) 23.1 0.4 68 9.7 0.9 63 (*) 14.5 0.4 17 10.2 2.1 4 86.6 0.5
0.25 mg/plate - 14 33 0.4 64 11.1 0.8 126 (*) 22.5 0.7 7 47.9 0.9 11 42.3 1.4
0.13 mg/plate - 23 22.2 0.6 61 9.1 0.8 120 2.9 0.7 8 6.9 1.0 7 34.3 1.0
0.06 mg/plate - 22 15.7 0.6 65 12.3 0.9 142 12 0.8 8 6.89 1.0 12 22 1.6
0.03 mg/plate - 26 9.8 0.7 71 11.4 0.9 171 3.2 1.0 11 10.8 1.3 13 7.7 1.7
                                 
Negative control + 27 20 - 74 4.1 - 272 4.3 - 10 24 - 8 15 -
Solvent control + 22 16 - 72 9.5 - 224 15 - 10 11 - 10 39.4 -
Positive control (2-AA) + 127 77.1 5.7 628 7.1 8.7 483 2.5 2.2 118 4.7 11.3 228 12.7 23.8
Positive control (B-a-P) + 625 1.3 27.5                        
5 mg/plate + 16 22.5 0.7 46 7 0.6 164 18.1 0.7 8 74.2 0.7 4 31.5 0.4
2.5 mg/plate + 34 28.1 1.5 53 5.4 0.7 307 8.3 1.4 7 15.7 0.7 6 0 0.6
1.25 mg/plate + 25 14.4 1.1 61 12.7 0.8 318 6.9 1.4 8 50 0.8 5 20 0.5
0.63 mg/plate + 26 17.6 1.2 75 21.4 1.0 290 3.8 1.3 7 7.9 0.7 9 11.1 0.9
0.31 mg/plate + 27 15.2 1.2 67 2.3 0.9 296 3.3 1.3 8 15.1 0.7 9 34.4 1.0
0.16 mg/plate + 36 26 1.6 70 5.2 1.0 301 3 1.3 8 37.5 0.8 8 12.5 0.8
0.08 mg/plate +                         12 17.8 1.2
RSD = relative standard deviation   IR = Induction Rate   * = cytotoxic effects (background growth)
Table 3: Mean values of the number of revertants and the induction factors obtained in the 3rd experiment 
Concentration S9 mix TA 1535
    mean RSD IR
Negative control - 12 38.9 -
Solvent control - 12 31.2 -
Positive control - 182 7.6 15.2
0.86 mg/plate - 9 41.6 0.7
0.72 mg/plate - 8 42.2 0.6
0.60 mg/plate - 8 36.7 0.7
0.50 mg/plate - 10 19.9 0.9
0.42 mg/plate - 10 33 0.9
0.35 mg/plate - 10 14.5 0.9
0.29 mg/plate - 11 13.7 0.9

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

According to the results achieved the test item Benzene, mono C10-13-alkyl derivs, distn. residues, sulfonated was evaluated as not mutagneic under the test conditions applied up to a test concentration of 5 mg per plate (related to the sulfonated material of 73.2%).
Executive summary:

This Bacterial Reverse Mutation Test (Ames test) with S. typhimurium strains TA 98, TA 100, TA 102 , TA 1535 and TA 1537 was carried out according to OECD Guideline 471 (July 1997).

The test item Benzene, mono C10 -13 -alkyl derivs, distn. residues, sulfonated was evaluated as not mutagenic under the test conditions applied up to a test concentration of 5 mg per plate (related to the sulfonated material of 73.2%) which is the highest test concentration required according to the guideline.

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