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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Mar - 27 Apr 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Ministerium fuer Umwelt und Verkehr Baden-Württemberg, Stuttgart
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Phenethyl cinnamate
EC Number:
203-120-3
EC Name:
Phenethyl cinnamate
Cas Number:
103-53-7
Molecular formula:
C17H16O2
IUPAC Name:
phenethyl cinnamate

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
- Source: Dr. Bruce N. Ames, University of California, Berkeley, California, U.S.A.
- Type and identity of media: Vogel-Bonner minimal medium plates enriched with histidine (260µM) and biotin (3μM). Ampicillin (25 µg per mL medium of the plate) is added to the plates used for the strains with the R-factor.
- complete medium plates: agar, 1.5%; Difco bacto nutrient broth, 0.8%; NaCL, 0.5%
- minimal agar plates: 20 to 25 mL of 1.5 % agar in Vogel-Bonner medium E with 2 % glucose
- Properly maintained: yes
- The Vogel-Bonner minimal plates, the test compound, and the S9-mix were checked for sterility.
Additional strain / cell type characteristics:
other: All strains contain an additional mutation: rfa. except TA102, the latter contains an uvrB-mutation. Strains TA98 & TA100 were developed by transferring a resistance factor (ampicillin) carried on plasmid pKM101, to strains TA1538 & TA1535 respectively.
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate fractions (S9)
Test concentrations with justification for top dose:
0, 15, 50, 150, 500, 1500 and 5000 µg/plate in the presence of S9
0, 5, 15, 50, 150, 1500 and 5000 μg/plate in the absence of S9
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: Standard plate-incorporation assay +/- liver homogenate activation

DURATION
- Preincubation period: at 37°C for 14-15 hours to a density of 0.5-3 x 109 cells per mL
- Exposure duration: inoculation overnight
- Expression/Selection time (cells in growth medium): 48 to 72 h at 37°C in the dark

NUMBER OF REPLICATIONS: 3 for each experimental point. The experiment was repeated in full after an intervall of at least 3 days.

DETERMINATION OF CYTOTOXICITY
- counting # revertant colonies (his+revertants)
- normal background lawn and/or precipitates
- microscopically: microcolony growth.
- reduction # revertant colonies and/or a diminution of the background lawn was taken as indication of bacteriotoxicity
- if there was doubt about the nature of colonies (revertants) or if positive mutagenic results are obtained, the genotype of revertant colonies are spot-checked

POSITIVE CONTROLS
In the absence of S9 mix:
- 0.7 µg/plate of sodium azide for strains TA100 and TA1535
- 2.5 µg/plate of 2-nitrofluorene for strain TA98
- 50 µg/plate of 9-aminoacridine for strain TA1537
- 0.15 µg/plate of mitomycin C for strain TA102

In the presence of SG9 mix:
- 0.8 µg/plate of 2-aminoanthracene for TA98 and TA100
- 0.9 µg/plate of 2-aminoanthracene for TA1535
- 1.0 µg/plate of 2-aminoanthracene for TA102
- 1.7 µg/plate of 2-aminoanthracene for TA1537
Statistics:
X2-test (Mohn and Ellenberger, 1977)

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Precipitation of the test compound on the plates was observed at 1500 and 5000 µg/plate.
The test compound was bacteriotoxic, but failed to induce a significant increase in the mutation frequency of the tester strains in the absence and presence of a metabolic activation system. Similarly, the estimation of the statistical significance of the differente between the mean number of revertants in the negative controls and the plates at each dosage level did not reveal a significant effect at any one of the test points.

Any other information on results incl. tables

Overview of individual data.
Legend:

p = precipitation

T = baceteriotoxic

Individual data experiment I, without S9

      Strain TA 98 Strain TA 100 Strain TA 102 Strain TA 1535 Strain 1537
Substance  Conc.
(µg/plate)
S9 # Mean SD # Mean SD # Mean SD # Mean SD # Mean SD
control 0 - 29 35 5 65 79 11 211 260 35 12 10 2 8 11 4
      37 86 283 10 11
      39 87 285 9 15
solvent control 0 - 30 32 3 78 81 3 240 237 3 11 10 2 11 11 1
      34 83 234 9 11
      32 83 238 11 12
test item 15 -       82 80 3 229 256 20 8 10 3      
        78 269 12  
        79 271 10  
test item 50 - 32 39 8 78 83 5 220 231 11 15 14 3 9 9 2
      36 83 228 16 11
      48 88 245 12 8
test item 150 - 37 34 5 70 72 2 196 199 15 19 15 4 7 8 3
      37 72 217 16 11
      29 73 184 11 6
test item 500 - 49 37 10 82 86 6 204 204 16 10 12 6 5 7 2
      26 84 185 6 7
      35 93 222 19 8
test item 1500 p - 44 36 7 92 86 8 196 174 T 18 9 8 2 9 8 2
      35 90 156 8 9
      29 77 170 7 7
test item 5000 p - 20 26 6                   7 6 T 2
      28       4
      31       7
NaN3 0.7 -       430 443 15       394 471 65      
        437   550  
        463   468  
2-NF 2.5 - 506 549 41                        
      602        
      538        
9-AA 50 -                         161 180 21
              207
              171
Mitocyin C 0.15 -             918 854 46            
          826    
          818    

Individual data experiment I, with S9

      Strain TA 98 Strain TA 100 Strain TA 102 Strain TA 1535 Strain 1537
Substance  Conc.
(µg/plate)
S9 # Mean SD # Mean SD # Mean SD # Mean SD # Mean SD
control 0 + 30 27 3 122 123 13 273 320 34 8 11 3 12 10 3
      26 109 342 12 10
      26 139 344 13 8
solvent control 0 + 17 21 4 118 117 5 292 299 6 16 13 4 13 14 4
      22 112 304 9 18
      25 122 302 13 11
test item 50 + 19 22 3 87 103 13 263 292 27 14 11 3 13 13 2
      22 116 287 8 14
      25 105 325 12 12
test item 150 + 27 26 6 135 120 14 293 311 14 8 10 2 13 13 3
      19 103 319 11 11
      31 121 322 10 16
test item 500 + 22 22 5 93 97 4 290 295 7 9 9 1 14 12 4
      26 99 303 9 8
      17 99 291 8 15
test item 1500 p + 22 19 5 109 106 3 235 259 42 7 11 4 13 13 2
      22 106 316 12 12
      13 104 225 14 15
test item 5000 p + 16 19 3 104 100 9 175 166 T 24 10 10 4 12 9 T 3
      22 89 188 14 7
      20 107 135 7 8
2-AA 0.8 + 800 799 40 1097 1018 60                  
      750 954      
      846 1002      
2-AA 0.9 +                   90 91 4      
            88  
            95  
2-AA 1.0 +             550 592 81            
          704    
          552    
2-AA 1.7 +                         164 203 36
              249
              195

Individual data experiment II, without S9

      Strain TA 98 Strain TA 100 Strain TA 102 Strain TA 1535 Strain 1537
Substance  Conc.
(µg/plate)
S9 # Mean SD # Mean SD # Mean SD # Mean SD # Mean SD
control 0 - 21 31 8 141 142 17 259 274 12 13 12 2 6 8 3
      37 161 277 11 10
      36 123 286 11 8
solvent control 0 - 33 31 4 135 118 14 235 264 23 14 13 2 9 8 2
      27 104 268 12 9
      32 114 289 14 6
test item 5 -             246 259 14            
          254    
          277    
test item 15 - 33 33 2       247 242 6       7 8 2
      35   243   9
      32   235   7
test item 50 - 44 39 5 145 131 16 238 255 34 5 8 3 6 9 4
      34 138 226 9 13
      40 111 302 11 8
test item 150 - 32 35 7 126 121 4 233 226 13 8 8 4 12 12 3
      43 120 235 4 9
      29 118 209 12 14
test item 500 - 22 29 6 112 115 3 239 203 28 9 8 2 10 9 4
      34 115 196 9 12
      32 117 175 6 5
test item 1500 p   25 34 9 114 115 3       8 11 5 8 8 1
      44 112   16 8
      34 118   9 9
test item 5000 p -       105 111 6       10 10 2      
        118   11  
        110   9  
NaN3 0.7 -       417 489 61       464 486 22      
        486   480  
        564   514  
2-NF 2.5 - 580 598 17                        
      596        
      618        
9-AA 50 -                         132 121 13
              126
              105
Mitocyin C 0.15 -             696 722 76            
          824    
          646    

Individual data experiment II, with S9

      Strain TA 98 Strain TA 100 Strain TA 102 Strain TA 1535 Strain 1537
Substance  Conc.
(µg/plate)
S9 # Mean SD # Mean SD # Mean SD # Mean SD # Mean SD
control 0 + 25 26 2 153 148 6 339 362 18 10 12 8 14 14 6
      25 141 369 22 20
      28 149 379 5 8
solvent control 0 + 21 23 3 153 133 20 318 348 22 12 12 4 12 13 2
      26 107 365 9 15
      21 139 362 16 13
test item 15 +             327 296 30       10 13 3
          258   14
          302   14
test item 50 + 19 20 2 125 124 10 318 302 13 10 9 2 17 16 2
      19 134 289 7 16
      21 113 298 10 15
test item 150 + 23 22 1 127 121 5 304 299 25 10 13 5 10 12 3
      22 117 325 11 14
      22 119 268 18 11
test item 500 + 19 16 3 111 124 11 321 309 26 13 12 2 13 14 4
      14 134 274 11 18
      16 128 331 13 12
test item 1500 p + 17 16 2 81 99 14 288 309 28 13 14 2 9 10 2
      17 108 222 13 12
      15 109 251 15 9
test item 5000 p + 17 13 4 100 99 2   254   8 9 2      
      9 98   8  
      14 98   11  
2-AA 0.8 + 968 1018 40 1299 1291 34                  
      1064 1327      
      1022 1248      
2-AA 0.9 +                   300 280 19      
            284  
            256  
2-AA 1.0 +             593 570 17            
          562    
          556    
2-AA 1.7 +                         294 245 45
              188
              252

Applicant's summary and conclusion

Conclusions:
The test item was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system, under the experimental conditions described.
Executive summary:

In the current study the mutagenicity of the substance was studied with 5 mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100, and TA102). The investigations were carried using the standard plate incorporation assay with and without liver homogenate (S9) as metabolic activation system. The test item was dissolved in DMSO and tested in concentrations of 15 to 5000 µg/plate in the presence and of 5 to 5000 µg/plate in the absence of S9.

In the absence of S9-mix the test substance was bacteriotoxic towards the strains TA102 at 1500 µg/plate and towards the strains TA1537 at 5000 µg/plate. In the presence of S9-mix the test item was bacteriotoxic towards the strains TA102 and TA1537 at 5000 µg/plate. Precipitation of the test compound an the plates was observed at 1500 and 5000 µg/plate.

Sodium azide, 2-nitrofluorene, 9-aminoacridine, mitomycin C, and 2-aminoanthracene served as positive controls and confirmed the reversion properties and the specificity of the bacterial strains as well as the efficacy of the metabolizing system.

In the concentration range investigated, the test substance did not induce a significant increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system.

 

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