Registration Dossier

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Diss Factsheets

Administrative data

Endpoint:
genetic toxicity in vitro
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Annex VII Section 8.4 of the REACH regulations requires the conduct of an appropriate in vitro gene mutation study. The OECD471 reverse mutation assay in bacteria (Ames test) is considered to complete fulfil the requirements for this assessment of mutagenicity

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Methyl 4(or 1)-isopropyl-1(or 4)-methylbicyclo[2.2.2]oct-5-ene-2-carboxylate
EC Number:
273-453-7
EC Name:
Methyl 4(or 1)-isopropyl-1(or 4)-methylbicyclo[2.2.2]oct-5-ene-2-carboxylate
Cas Number:
68966-86-9
Molecular formula:
C14H22O2
IUPAC Name:
Methyl 4(or 1)-isopropyl-1(or 4)-methylbicyclo[2.2.2]oct-5-ene-2-carboxylate

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 Liver homogenates from Spargue-Dawley male rats with Aroclor 1254
Test concentrations with justification for top dose:
Presence of S9; 15, 50, 150, 500 and 1500 μg/plate
Absence of S9; 15, 50, 150, 500 and 1500 μg/plate
Top dose based on results of the initial toxicity test
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Not reported
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
Remarks:
Experiment AM002031 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
Experimeny AM002031 with metabolic activation and Experiment AM002032 with metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
mitomycin C
Remarks:
Experiment AM002032 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
Remarks:
Experiment AM002033 with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): 0.1 mL bacteria

DURATION
- Preincubation period: 11 - 12 hours
- Exposure duration: 48 to 72 hours
- Expression time (cells in growth medium): 48 to 72 hours
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable

NUMBER OF REPLICATIONS: 3

Plates were kept in the dark for 48 to 72 hours at 37.5 ºC

NUMBER OF CELLS EVALUATED: 1.3 x 10^9 to 2.7 x 10^9

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Micrscopic evaluation of revertants
- Any supplementary information relevant to cytotoxicity: None reported

OTHER EXAMINATIONS: None reported
Rationale for test conditions:
Following standard guidelines
Evaluation criteria:
The number of revertant colonies on the plates, with and without the test compound, were compared to evaluate mutagenicity. A reduction in the number of revertant colonies and/or a diminution of the background lawn was taken as an indication of bacteriotoxicity. If there was concern about the nature of colonies scored as revertants and when positive mutagenic results are obtained, the genotype of revertant colonies were spot-checked by picking and streaking on histidine free plates.
Statistics:
None reported

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Not mutagenic
Cytotoxicity / choice of top concentrations:
not specified
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
Remarks:
Not mutagenic
Cytotoxicity / choice of top concentrations:
not specified
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
Remarks:
Not mutagenic
Cytotoxicity / choice of top concentrations:
not determined
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
Remarks:
Not mutagenic
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Not mutagenic
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None reported
- Effects of osmolality: None reported
- Evaporation from medium: None reported
- Water solubility: None reported
- Precipitation: None reported
- Definition of acceptable cells for analysis: Not reported
- Other confounding effects: None reported

RANGE-FINDING/SCREENING STUDIES: Performed but results not reported

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: Not applicable

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: 1.3 x 10^9 to 2.9 x 10^9
- Indication whether binucleate or mononucleate where appropriate: Not applicable

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: strain, mutagen (concentration of mutagen in μg/plate), mean of revertants per plate (minimum/maximum). The data of test without S9 are: TA1535, NaN3 (0.7), 348176 (208/659); TA1537, 9-AA (50), 265±106 (133/479); TA98, 2-NF (2.5), 406+95 (98/556); TA100, NaN3 (0.7), 379±57 (287/491); TA102, Mitotnycin C (0.15) 703±188 (369/997). The data of the tests with lot KH2202 of S9 are: TA1535, 2-AA (0.9), 130+34 (62/153); TA1537, 2-AA (1.7), 237148 (165/255); TA98, 2-AA (0.8), 624±138 (368/754); TA100, 2-AA (0.8), 600-1191 (202/764); TA102, 2-AA (0.9), 5341117 (349/636). Additional data of the tests with lot KI-12202 of S9 are: TA100, B(a)P (5.0), 719123 (701/752); TA102, B(a)P (5.0), 427119 (406/442).
- Negative (solvent/vehicle) historical control data: See table below

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Revertent colonies
- Other observations when applicable: None reported

Any other information on results incl. tables

Table 1: Induction of revertants in S. typhimurium by the test item in the absente of a metabolizing system. (Experiment AM00203 1)

 Substance  Concentration (μg/plate)  S9  Number of revertants per plate                       
 TA98  TA100  TA102  TA1535  TA1537
 Counts  Mean  SD  Counts  Mean  SD  Counts  Mean  SD  Counts  Mean  SD  Counts  Mean  SD
Control   0  -  49  55  6  101  110  8  203  234  23  19  13  6  4  7  3
       55      118      245      11      9    
       62      110      253      8      7    
 Solvent Control  0  -  58  57  7  97  98  2  252  256  6  15  13  3  9  10  2
       63      97      252      12      9    
       49      99      263      11      12    
 Test item  5  -        104  97  6        11  12  2  8  9  2
             93            14      11    
             93            11      8    
 Test item  15  -  44  48  4  106  104  3  239  234  11  16  13  3  12  9  3
       49      101      220      12      6    
       52      105      244      12      10    
 Test item  50  -  57  54  3  107  95  13  242  249  18  12  10  3  9  8  3
       51      99      272      9      5    
       55      79      232      8      10    
 Test item  150  -  33  32  3  84  95  9  230  227  8  11  9  2  4  4 T  1
       33      99      233      9      4    
       29      101      217      8      5    
 Test item  500  -  29  29 T  5  71  66 T  10  195  226  23  10  12  3  6  3 T  3
       25      74      236      12      3    
       34      54      246      15      1    
 Test item  1500  -  23  25 T  5        212  212  3            
       22            215                
       30            210                
 NaN3  0.7  -        341  369  21        372  404  23      
             380            421          
             385            418          
 2 -NF  2.5  -  410  428  14                        
       433                            
       440                            
 9 -AA  50  -                          283  247  85
                               327    
                               131    
 Mitomycin C  0.15  -              534  604  53            
                   658                
                   620                

- : without S9-mix; SD: standard deviation; T: bacteriotoxic; solvent control: DMSO, 50 pl/plate; 2-NF: 2-nitrofluorene; 9-AA: 9-aminoacridine;

titer of bacterial suspensions (viable cells/ml), TA98: 2.2x10^9 (232,262,178), TA100: 2.8x10^9 ( 262,304,264), TA102: 3.2x10^9 (364,280,310), TA1535: 2.9x10^9 (286,316,278), TA1537: 1.3x10^9 (128,138)

Table 2: Induction of revertants in S. typhimurium by Tricyclate in the presence of a metabolizing system. (Experiment AM002031)

 Substance  Concentration (μg/plate)  S9  Number of revertants per plate                       
 TA98  TA100  TA102  TA1535  TA1537
 Counts  Mean  SD  Counts  Mean  SD  Counts  Mean  SD  Counts  Mean  SD  Counts  Mean  SD
Control   0  +  32  37  7  112  109  6  249  262  10  11

 11

 3

 13

 11

 2

 

 

 

 34

 

 

 114

 

 

 268

 

 

 14

 

 

 10

 

 

 

 

 

 46

 

 

 102

 

 

 269

 

 

 9

 

 

 11

 

 

 Solvent Control

 0

 +

 35

 42

 8

 103

 103

 0

 276

 270

 6

 9

 10

 2

 13

 12

 3

 

 

 

 52

 

 

 103

 

 

 264

 

 

 9

 

 

 10

 

 

 

 

 

 39

 

 

 103

 

 

 270

 

 

 11

 

 

 14

 

 

 Test item

 15

 +

 48

 45

 7

 100

 98

 5

 228

 244

 14

 10

 9

 1

 14

 12

 3

 

 

 

 37

 

 

 101

 

 

 246

 

 

 9

 

 

 12

 

 

 

 

 

 50

 

 

 92

 

 

 259

 

 

 9

 

 

 10

 

 

 Test item

 50

 +

 38

 39

 2

 89

 102

 11

 238

 259

 16

 9

 10

 3

 15

 16

 3

 

 

 

 40

 

 

 113

 

 

 271

 

 

 13

 

 

 18

 

 

 

 

 

 38

 

 

 105

 

 

 267

 

 

 9

 

 

 14

 

 

 Test item

 150

 +

 37

 35

 3

 93

 97

 5

 247

 227

 19

 10

 8

 3

 10

 12

 4

 

 

 

 32

 

 

 95

 

 

 229

 

 

 5

 

 

 10

 

 

 

 

 

 36

 

 

 103

 

 

 204

 

 

 9

 

 

 16

 

 

 Test item

 500

 +

 38

 35

 3

 59

 73

 11

 238

 240

 3

 5

 5

 2

 10

 6

 3

 

 

 

 34

 

 

 82

 

 

 239

 

 

 4

 

 

 6

 

 

 

 

 

 32

 

 

 77

 

 

 242

 

 

 6

 

 

 8

 

 

 Test item

 1500

 +

 34

 34

 5

 25

 27 T

 4

 209

 208

 10

 7

 8

 1

 9

 6 T

 5

 

 

 

 40

 

 

 32

 

 

 219

 

 

 8

 

 

 8

 

 

 

 

 

 29

 

 

 25

 

 

 197

 

 

 8

 

 

 1

 

 

 2-AA

 0.8

 +

 588

 643

 45

 872

 859

 10

 

 

 

 

 

 

 

 

 

 

 

 

 644

 

 

 854

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 696

 

 

 852

 

 

 

 

 

 

 

 

 

 

 

 2 -AA

 0.9

 +

 

 

 

 

 

 

 415

 397

 16

 141

 160

 15

 

 

 

 

 

 

 

 

 

 

 

 

 378

 

 

 167

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 398

 

 

 172

 

 

 

 

 

 2 -AA

 1.7

 +

 

 

 

 

 

 

 

 

 

 

 

 

 272

 302

 22

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 318

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 315

 

 

+: with S9-mix: SD: standard deviation; T: bacteriotoxic; solvent control: DMSO, 50 pliplate; 2-AA: 2-aminoanthracene; titer of bacterial suspensions (viable cells/m1), TA98: 2.2x10^9 (232,262,178), TA100: 2.8x10^9 (262,304,264), TA102: 3.2x10^9 (364,280,310), TA1535: 2.9x1^9 (286,316,278), TA1537: 1.3x10^9 ( 128,138)

Table 3: Induction of revertants in S. typhimurium by the test item in the absente of a metabolizing system. (Experiment AM002032)

Substance  Concentration (μg/plate)    Number of revertants per plate
 TA100  TA102  TA1535
 Counts  Mean  SD  Counts  Mean  SD  Counts  Mean  SD
 Control  0  94  101  8  220  255  26  20  15  5
    98       267      10    
     111      279      14    
 Solvent control  0  108  101  6  248  255  6  11  15  4
     100      256      19    
     96      261      15    
 Test item  5  81  92  10            
     92                
     102                
 Test item  15  99  89  8        15  12  3
     81            10    
     88            10    
 Test item  50  109  94  12  238  241  5  9  11  3
     84      247      11    
     89      239      14    
 Test item  150  84  92  8  252  255  17  15  14  3
     91      236      15    
     102      276      11    
 Test item  500  61  53 T  7  222  198  22  9  13  4
     46      202      15    
     51      171      14    
 Test item  1500        174  167 T  6  9  10  1
           165      10    
           162      10    
 Test item  5000        133  140 T  21  5  6 T  2
           168      5    
           120      7    
 NaN3  0.7  398  354  33        346  387  31
     323            398    
     341            417    
 Mitomycin C  0.15        659  743  61      
           774          
           796          

SD: standard deviation; T: bacteriotoxic; solvent control: DMSO, 50 ul/plate; titer of bacterial suspensions (viable cells/ml), TA100: 2.7x10^9 ( 230,298,270), TA102: 2.5x10^9 ( 243,279,229), TA1535: 2.3x10^9 ( 209,244,235)

Table 3a: Induction of revertants in S. typhimurium by the test item in the absente of a metabolizing system. (Experiment AM002033)

Substance  Concentration (μg/plate)   Number of revertants per plate
 TA98  TA1537
 Counts  Mean  SD  Counts  Mean  SD
 Control  0  33  44  9  14  12  2
     48      12    
     52      11    
 Solvent control  0  46  50  6  10  11  2
     57      12    
     48      11    
 Test item  1.5  52  49  5  11  11  1
     52      12    
     43      11    
 Test item  5  47  46  4  7  10  5
     48      15    
     42      8    
 Test item  15  40  38  4  11  13  3
     40      11    
     33      16    
 Test item  50  33  37  5  9  10  3
     36      9    
     42      13    
 Test item  150  38  31 T  7  8  5 T  3
     30      6    
     24      2    
 2 -NF  2.5  386  378  22      
     349          
     399          
 2 -NF  50        176  174  23
           200    
           147    

SD: standard deviation; T: bacteriotoxic; solvent control: DMSO, 50 ul/plate; 2-NF: 2-nitrofluorene; 9-AA: 9-aminoacridine; titer of bacterial suspensions (viable cells/ml), TA98: 2.7x10^9 ( 245,298,273), TA1537: 2.5x10^9 (196,308,257)

Table 4: Induction of revertants in S. typhimurium by Tricyclate in the presence of a metabolizing system. (Experiment AM002032)

Substance       Concentration (μg/plate)  S9  Number of revertants per plate
 TA98  TA100  TA102  TA1535   TA1537
 Counts  Mean  SD  Counts  Mean  SD  Counts  Mean  SD  Counts  Mean  SD  Counts  Mean  SD
 Control  0  +  43  37  6  104  109  11  237  252  16  9  8  2  11  13  3
       32      100      272      7      12    
       36      122      247      7      16    
 Solvent Control  0  +  32  35  6  95  97  3  276  273  12  7  9  4  6  11  5
       42      95      285      8      17    
       31      100      259      13      11    
 Test item  5  +        89  92  3              13  16  3
             93                  16    
             94                  18    
 Test item  15  +        107  102  8              10  12  2
             106                  13    
             92                  12    
 Test item  50  +  33  38 4 87  88  3  233  250  18  18  11  6  10  15  5
       40      91      243      8      17    
       40      87      273      8      18    
 Test item  150  +  34  34  4  110  108  3  270  261  23  7  10  4  14  12  3
       37      109      231      9      13    
       30      105      282      14      10    
 Test item  500  +  31  31  4  78  70  15  260  243  24  11  10  3  12  9  3
       27      81      258      8      9    
       34      50      211      12      6    
 Test item  1500  +  25  29  4        210  206  5  6  6 T  1      
       29            207      6          
       33            200      7          
 Test item  5000  +  31  27 T  6        161  135 T  20  5  5 T  2      
       30            127      4          
       20            116      6          
 2 -AA  0.8  +  585  541  34  976  911  60                  
       531      834                      
       507      924                      
 2 -AA  0.9  +              591  658  49  158  171  13      
                   684      187          
                   699      168          
 2 -AA  1.7  +                          217  246  34
                               292    
                               229    

+: with S9-mix: SD: standard deviation; T: bacteriotoxic; solvent control: DMSO, 50 ul/plate; 2-AA: 2-aminoanthracene; titer of bacterial suspensions (viable cells/ml), TA98: 2.7x10&9 ( 245,298,273), TA100: 2.7x10^9 ( 230,298,270), TA102: 2.5x10^9 ( 243,279,229), TA1535: 2.3x10^9 ( 209,244,235), TA1537: 2.5x10^9 ( 196,308,257)

Table 5: Historic negative control data

Strain  -S9  +S9
 Spontaneous Solvent control  Spontaneous  Solvent control
 TA1535  14 ± 6 (10/34)  14 ± 4 (9/24)  14 ± 5 (8/19)  13 ± 4 (8/21)
 TA1537  11 ± 4 (7/18)  11 ± 3 (6/17)  14 ± 3 (11/21)  15 ± 4 (11/26)
 TA98  33 ± 11 (21/52)  31 ± 11 (19/60)  35 ± 11 (16/64)  36 ± 11 (20/57)
 TA100  106 ± 21 (82/147)  101 ± 19 (81/132)  103 ± 15 (85/130)  102 ± 13 (87/122)
 TA102  249 ± 29 (198/291)  251 ± 19 (219/289)  273 ± 15 (234/330)  275 ± 28 (238/345)

Applicant's summary and conclusion

Conclusions:
In the concentration range investigated, the test item did not induce a significant increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system. Therefore under the experimental conditions 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.
Executive summary:

The test item was investigated in an Ames test with Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system at a range of concentrations of 5 to 5000 μg/plate following the OECD 471 guideline. In the concentration range investigated, the test item did not induce a significant increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system. Therefore under the experimental conditions 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.

The test is a GLP compliant, guideline experimental study available as an unpublished study reported, deemed reliable without restrictions, and therefore adequate for use as a supporting study.