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Toxicological information

Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD) acc. to GLP, with limitations acc. to current standart protocols
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD) acc. to GLP, with limitations acc. to current standart protocols
Justification for type of information:
ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source substances and target substance have similar physical-chemical properties and (eco)toxicological properties because they are either stereoisomers of the target substance, are hydrolysed to the same substance or their chemical structure differs only by an additional double bond. This prediction is supported by data on the substances themselves.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance, L-Citronellol, is a mono-constituent substance (EC No. 231-415-7, CAS no. 7540-51-4 consisting of a C8 carbon backbone, methyl substituents at C3 and C7, one double bond and a hydroxyl group. The substance is optically active, comprising a single, pure enantiomeric laevo form.

The source substance, DL-Citronellol, is a mono-constituent substance (EC No. 203-375-0, CAS no. 106-22-9, consisting of a C8 carbon backbone, methyl substituents at C3 and C7, one double bond and a hydroxyl group. The substance is an equimolar mixture of two optical isomers (enantiomers).

The source substance, citronellyl acetate, is a mono-constituent substance (EC No. 205-775-0, CAS no. 150-84-5) consisting of a C8 carbon backbone, methyl substituents at C3 and C7, one double bond and an acetate group.

The source substance, geraniol and it’s isomer, consist of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group. The only difference between the isomers is the position of the first double bond.

The source substance, geraniol and nerol, is a multi-constituent substance of E/Z isomers (EC No. 906-125-5). The constituents consist of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group.

The source substance, geraniol, is a mono-constituent substance (EC No. 203-377-1, CAS no. 106-24-1), consisting of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group. Geraniol is a pure form of the E-isomer.

The source substance, nerol, is a mono-constituent substance (EC No. 203-378-7, CAS no. 106-25-2), consisting of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group. Nerol is a pure form of the Z-isomer.
The source and target substances are both of high purity with a low concentration of impurities.

3. ANALOGUE APPROACH JUSTIFICATION
The read across hypothesis is based on structural similarity where the source substances only differ in the enantiomeric ratio or an additional double bond. Another source substance is expected to be hydrolysed to the same structure as the target substance.
In a non-chiral environment the target and source chemical DL-Citronellol will have identical properties, but in the chiral environment of living organisms the enantiomers may possess different carcinogenicity and teratogenicity (in a chiral environment, stereoisomers might experience selective absorption, protein binding, transport, enzyme interactions and metabolism, receptor interactions, and DNA binding). All endpoints read-across from DL-Citronellol are considered to be acceptable for this substance assuming that 50% of the target compound is available in the test material.
The source substance citronellyl acetate is read-across from as part of a weight of evidence approach in the repeated dose toxicity endpoint. As this substance is hydrolysed to Citronellol within 2 hours, this read-across endpoint is acceptable in the weight of evidence approach used.
The source substances geraniol, nerol and the reaction mass of geraniol/nerol differ from the target substance only by an additional double bond at C2. These structures are considered to represent a worst case scenario due to the additional potential reactive feature of the second double bond. The genotoxicity, repeated dose and reproductive toxicity endpoints read-across from these substances are therefore acceptable as a worst case assumption.

4. DATA MATRIX
Please refer to the data matrix included in the read-across justification document attached in Section 13.2.
Reason / purpose:
read-across source
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
analytical investigations (stability of the test substance in the carrier) were not carried out; only 4 strains tested
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
EEC Directive 84/449, B14
GLP compliance:
yes
Remarks:
Department of Toxicology, BASF AG
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
His operon
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction from Aroclor induced Sprague-Dawley rats
Test concentrations with justification for top dose:
0.8 - 5000 µg/plate - SPT: TA100, TA98;
0.8 - 500 µg/plate - SPT: TA1535, TA1537, PIT: all tester strains
SPT = standard plate test
PIT = preincubation test
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: complete solubility of the test substance in DMSO
Negative controls:
yes
Solvent controls:
yes
True negative controls:
yes
Remarks:
sterility control
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 10 µg/plate in DMSO; TA 1535, TA 1537, TA 98 and TA 100
Remarks:
with metabolic activation
Negative controls:
yes
Solvent controls:
yes
True negative controls:
yes
Remarks:
sterility control
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without metabolic activation Migrated to IUCLID6: 5 µg/plate, in DMSO; TA 100 and TA 1535
Negative controls:
yes
Solvent controls:
yes
True negative controls:
yes
Remarks:
sterility control
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylendiamine; 10 µg, in DMSO; TA 98
Remarks:
without metabolic activation
Negative controls:
yes
Solvent controls:
yes
True negative controls:
yes
Remarks:
sterility control
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation Migrated to IUCLID6: 100 µg/plate, in DMSO; TA 1537
Details on test system and conditions:
positive control substances:
2-AA: 2-aminoanthracene
MNNG: N-methyl-N'-nitro-N-nitroso-guanidine
NPD: 4-nitro-o-phenylendiamine
AAC: 9-aminoacridine

1st experiment:
An SPT with TA 98 and TA 100 showed, that citronellol conentrations >500 µg/plate were cytotoxic.
2nd experiment:
In the second STP with all tester strains concentrations were adapted with 500 µg/plate being the highest concentration.
3rd experiment:
An PIT with all strains and the adapted concentrations was performed. Since in the second SPT colony scoring of TA 98 was not possible due to contamination, the SPT with this strain was repeated additional to the PIT.
Evaluation criteria:
In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
other: bacteriotoxic effect at doses >= 500 µg/plate
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'.

Tabel 1: Standard Plate Test, Strain TA 100

 

revertants / plate

titer dil.

quotient

dose [µg/plate] -S9 M SD +S9* M SD exp-6 -S9

+S9*

 neg. control DMSO 129 126 7 106 118 10 20 1.0 1.0
  131     125     30    
  118     123     11    
 20 143 131 10 156 137 20   1.0 1.2
  127     116          
  124     140          
 100 102 114 12 116 134 15   0.9 1.1
  115     141          
  126     144          
 500 B - - 57 73 14   - 0.6
  B     80          
  B     81          
 2500 0 0 0 0 0 0 0 0.0 0.0
  0     0     0    
  0     0     0    
 5000 0 0 0 0 0 0 0 0.0 0.0
  0     0          
  0     0          
 pos. control 2 -AA [10]       1900 1723 204     14.6
        1500          
        1770          
 pos. control MNNG [5] 1620 1523 84         12.1  
  1480                
  1470                

*: S9 -fraction/cofactors = 1:9;       B: reduced his- background

Table 2: Standard Plate Test, Strain TA 98

 

revertants / plate

titer dil.

quotient

dose [µg/plate] -S9 M SD +S9* M SD exp-6 -S9

+S9*

 neg. control DMSO 23 25 2 40 41 2 31 1.0 1.0
  25     41     20    
  26     43     21    
 20 26 25  46  47    1.0 1.1
  24      47           
  25      48          
 100 21 23  46 36 12    0.9 0.9
  28     38          
  19      23           
 500 15 17 2 18 17 4   0.7 0.4
  18     13          
  17     20          
 2500 0 0 0 0 0 0 0 0.0 0.0
  0     0     0    
  0     0     0    
 5000 0 0 0 0  0  0 0 0.0  0.0
  0     0          
  0     0          
 pos. control 2 -AA [10]       1530 1607 108     38.9
        1730          
        1560          
 pos. control NPD [10] 1250 1243 140         50.4  
  1380                
  1100                

*: S9 -fraction/cofactors = 1:9

Table 3: Standard Plate Test, Strain TA 1535

 

revertants / plate

titer dil.

quotient

dose [µg/plate] -S9 M SD +S9* M SD exp-6 -S9

+S9*

 neg. control DMSO 16 14 2 12 16 6 16 1.0 1.0
  13     13     11    
  13     23     16    
0.8 12 16 4 17 15 2   1.2 1.0
  17     16          
  20     13          
4 19 18 4 8 13 4   1.3 0.8
  14     16          
  22     15          
20 20 20 2 12 13 1   1.4 0.8
  18     13          
  21     14          
100 21 20 1 8 17 8 22 1.4 1.0
  19     22     21    
  19     20     20    
500 11 13 3 12 14 2 15 0.9 0.9
  11     15     11    
  16     14     12    
 pos. control 2 -AA [10]       160 200 67     12.5
        162          
        277          
 pos. control MNNG [5] 1250 1260 10         90.0  
  1270                
  1260                

*: S9 -fraction/cofactors = 1:9

Table 4: Standard Plate Test, Strain TA 100

 

revertants / plate

titer dil.

quotient

dose [µg/plate] -S9 M SD +S9* M SD exp-6 -S9

+S9*

 neg. control DMSO 111 106 6 132 129 3 20 1.0 1.0
  109     127     11    
  99     129     35    
0.8 94 97 3 144 137 6   0.9 1.1
  100     133          
  97     133          
4 103 115 11 115 114 11   1.1 0.9
  124     125          
  119     103          
20 117 114 6 149 126 21   1.1 1.0
  107     120          
  118     109          
100 107 112 6 108 129 18 14 1.1 1.0
  109     135     18    
  119     143     12    
500 120 108 12 120 109 12 14 1.0  0.8
  97     97     10    
  107     110     10    
 pos. control 2 -AA [10]       1010 1105 85     8.5
        1130          
        1175          
 pos. control MNNG [5] 1340 1197 127         11.3  
  1150                
  1100                

*: S9 -fraction/cofactors = 1:9

Table 5: Standard Plate Test, Strain TA 1537

 

revertants / plate

titer dil.

quotient

dose [µg/plate] -S9 M SD +S9* M SD exp-6 -S9

+S9*

 neg. control DMSO 19 11 7 9 12 2 14 1.0 1.0
  7     13     18    
  8     13     19    
0.8 8 8 1 16 12 5   0.7 1.1
  9     14          
  7     7          
4 11 9 6 11 10 2   0.8 0.8
  14     10          
  2     8          
20 7 8 3 14 13 2   0.7 1.1
  5     10          
  11     14          
100 9 11 2 14 11 3 5 1.0 0.9
  13     10     3    
  11     8     7    
500 13 12 2 9 8 1 6 1.0  0.7
  9     8     5    
  13     8     8    
 pos. control 2 -AA [10]       300 265 34     22.7
        233          
        261          
 pos. control AAC [100] 374 464 95         40.9  
  455                
  563                

*: S9 -fraction/cofactors = 1:9

Table 6: Preincubation Test, Strain TA 1535

 

revertants / plate

titer dil.

quotient

 dose [µg/plate] -S9 M SD +S9* M SD exp-6 -S9

+S9*

 neg. control DMSO 15 17 3 13 15 2 14 1.0 1.0
  16     16     16    
  21     16     10    
0.8 21 21 2 22 21 6   1.2 1.4
  22     15          
  19     26          
4 27 24 4 17 17 1   1.4 1.1
  25     17          
  19     16          
20 26 25 1 18 18 2   1.4 1.3
  24     21          
  24     19          
100 22 21 4 14 16 4 5 1.2 1.1
  17     14     8    
  24     21     2    
500 B 0 0 B - - 0 0.0 -
  0     B     0    
  0     B     0    
 pos. control 2 -AA [10]       134 170 33     11.4
        198          
        179          
 pos. control MNNG [5] 851 814 49         47.0  
  832                
  759                

*: S9 -fraction/cofactors = 1:9;       B: reduced his- background

Table 7: Preincubation Test, Strain TA 100

 

revertants / plate

titer dil.

quotient

 dose [µg/plate] -S9 M SD +S9* M SD exp-6 -S9

+S9*

 neg. control DMSO 112 11 6 102 105 5 27 1.0 1.0
  116     111     19    
  104     102     23    
0.8 90 96 15 122 121 7   0.9 1.1
  84     113          
  113     127          
4 116 117 11 159 150 8   1.1 1.4
  107     143          
  128     149          
20 129 127 14 111 108 4   1.1 1.0
  112     110          
  139     103          
100 113 124 9 118 110 9 3 1.1 1.1
  129     113     8    
  129     100     2    
500 0 0 0 B - - 0 0.0 -
  0     B     0    
  0     B     0    
 pos. control 2 -AA [10]       848 1133 341     10.8
        1040          
        1510          
 pos. control MNNG [5] 684 675 10         6.1  
  676                
  664                

*: S9 -fraction/cofactors = 1:9;       B: reduced his- background

Table 8: Preincubation Test, Strain TA 1537

 

revertants / plate

titer dil.

quotient

 dose [µg/plate] -S9 M SD +S9* M SD exp-6 -S9

+S9*

 neg. control DMSO 11 9 2 11 11 1 3 1.0 1.0
  7     12     2    
  10     10     4    
0.8 14 12 4 5 10 5   1.3 0.9
  7     14          
  14     10          
4 7 8 2 11 11 2   0.9 1.0
  7     9          
  10     13          
20 10 8 2 10 10 3   0.8 0.9
  7     13          
  6     7          
100 13 12 3 12 9 3 4 1.3 0.8
  14     7     3    
  8     8     6    
500 0 0 0 B - - 0 0.0 -
  0     B     0    
  0     B     0    
 pos. control 2 -AA [10]       119 116 9     10.5
        123          
        105          
 pos. control AAC [100] 1200 1197 55         128.2  
  1250                
  1140                

*: S9 -fraction/cofactors = 1:9;       B: reduced his- background

Table 9: Preincubation Test, Strain TA 98

 

revertants / plate

titer dil.

quotient

 dose [µg/plate] -S9 M SD +S9* M SD exp-6 -S9

+S9*

 neg. control DMSO 30 31 1 35 35 5 21 1.0 1.0
  32     31     15    
  30     40     23    
0.8 17 20 4 41 35 6   0.6 1.0
  24     30          
  18     35          
4 21 23 3 40 38 4   0.7 1.1
  21     33          
  26     40          
20 20 22 7 34 36 3   0.7 1.0
  16     40          
  29     35          
100 14 20 6 30 31 3 0 0.7 0.9
  24     28     0    
  23     34     0    
500 0 0 0 B - - 0 0.0 -
  0     B     0    
  0     B     0    
 pos. control 2 -AA [10]       1370 1437 59     40.7
        1480          
        1460          
 pos. control NPD [10] 1320 1197 120         39.0  
  1190                
  1080                

*: S9 -fraction/cofactors = 1:9;       B: reduced his- background

Table 10: Standard Plate Test, Strain TA 98

 

revertants / plate

titer dil.

quotient

 dose [µg/plate] -S9 M SD +S9* M SD exp-6 -S9

+S9*

 neg. control DMSO 21 22 3 39 38 4 21 1.0 1.0
  25     41     15    
  19     33     29    
0.8 19 24 5 37 39 2   1.1 1.0
  25     39          
  29     40          
4 26 27 1 31 33 2   1.2 0.9
  28     33          
  26     34          
20 25 28 7 34 31 3   1.3 0.8
  22     28          
  36     32          
100 25 26 1 28 29 2 11 1.2 0.8
  27     32     15    
  27     28     16    
500 24 24 5 28 28 0 14 1.1  0.7
  20     28     9    
  29     28     9    
 pos. control 2 -AA [10]       1260 1132 133     30.0
        995          
        1140          
 pos. control NPD [10] 825 794 35         36.6  
  801                
  756                

*: S9 -fraction/cofactors = 1:9

Conclusions:
Interpretation of results (migrated information):
negative

Data source

Reference
Reference Type:
other company data
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
analytical investigations (stability of the test substance in the carrier) were not carried out; only 4 strains tested
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
EEC Directive 84/449, B14
GLP compliance:
yes
Remarks:
Department of Toxicology, BASF AG
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Citronellol
- Physical state: colorless liquid
- Analytical purity: 95%
- Date of manufacturing: April 5, 1991
- Storage condition of test material: room temperature (under N2)

Method

Target gene:
His operon
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction from Aroclor induced Sprague-Dawley rats
Test concentrations with justification for top dose:
0.8 - 5000 µg/plate - SPT: TA100, TA98;
0.8 - 500 µg/plate - SPT: TA1535, TA1537, PIT: all tester strains
SPT = standard plate test
PIT = preincubation test
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: complete solubility of the test substance in DMSO
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
True negative controls:
yes
Remarks:
sterility control
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 10 µg/plate in DMSO; TA 1535, TA 1537, TA 98 and TA 100
Remarks:
with metabolic activation
Negative controls:
yes
Solvent controls:
yes
True negative controls:
yes
Remarks:
sterility control
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without metabolic activation Migrated to IUCLID6: 5 µg/plate, in DMSO; TA 100 and TA 1535
Negative controls:
yes
Solvent controls:
yes
True negative controls:
yes
Remarks:
sterility control
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylendiamine; 10 µg, in DMSO; TA 98
Remarks:
without metabolic activation
Negative controls:
yes
Solvent controls:
yes
True negative controls:
yes
Remarks:
sterility control
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation Migrated to IUCLID6: 100 µg/plate, in DMSO; TA 1537
Details on test system and conditions:
positive control substances:
2-AA: 2-aminoanthracene
MNNG: N-methyl-N'-nitro-N-nitroso-guanidine
NPD: 4-nitro-o-phenylendiamine
AAC: 9-aminoacridine

1st experiment:
An SPT with TA 98 and TA 100 showed, that citronellol conentrations >500 µg/plate were cytotoxic.
2nd experiment:
In the second STP with all tester strains concentrations were adapted with 500 µg/plate being the highest concentration.
3rd experiment:
An PIT with all strains and the adapted concentrations was performed. Since in the second SPT colony scoring of TA 98 was not possible due to contamination, the SPT with this strain was repeated additional to the PIT.
Evaluation criteria:
In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
other: bacteriotoxic effect at doses >= 500 µg/plate
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'.

Any other information on results incl. tables

Tabel 1: Standard Plate Test, Strain TA 100

 

revertants / plate

titer dil.

quotient

dose [µg/plate] -S9 M SD +S9* M SD exp-6 -S9

+S9*

 neg. control DMSO 129 126 7 106 118 10 20 1.0 1.0
  131     125     30    
  118     123     11    
 20 143 131 10 156 137 20   1.0 1.2
  127     116          
  124     140          
 100 102 114 12 116 134 15   0.9 1.1
  115     141          
  126     144          
 500 B - - 57 73 14   - 0.6
  B     80          
  B     81          
 2500 0 0 0 0 0 0 0 0.0 0.0
  0     0     0    
  0     0     0    
 5000 0 0 0 0 0 0 0 0.0 0.0
  0     0          
  0     0          
 pos. control 2 -AA [10]       1900 1723 204     14.6
        1500          
        1770          
 pos. control MNNG [5] 1620 1523 84         12.1  
  1480                
  1470                

*: S9 -fraction/cofactors = 1:9;       B: reduced his- background

Table 2: Standard Plate Test, Strain TA 98

 

revertants / plate

titer dil.

quotient

dose [µg/plate] -S9 M SD +S9* M SD exp-6 -S9

+S9*

 neg. control DMSO 23 25 2 40 41 2 31 1.0 1.0
  25     41     20    
  26     43     21    
 20 26 25  46  47    1.0 1.1
  24      47           
  25      48          
 100 21 23  46 36 12    0.9 0.9
  28     38          
  19      23           
 500 15 17 2 18 17 4   0.7 0.4
  18     13          
  17     20          
 2500 0 0 0 0 0 0 0 0.0 0.0
  0     0     0    
  0     0     0    
 5000 0 0 0 0  0  0 0 0.0  0.0
  0     0          
  0     0          
 pos. control 2 -AA [10]       1530 1607 108     38.9
        1730          
        1560          
 pos. control NPD [10] 1250 1243 140         50.4  
  1380                
  1100                

*: S9 -fraction/cofactors = 1:9

Table 3: Standard Plate Test, Strain TA 1535

 

revertants / plate

titer dil.

quotient

dose [µg/plate] -S9 M SD +S9* M SD exp-6 -S9

+S9*

 neg. control DMSO 16 14 2 12 16 6 16 1.0 1.0
  13     13     11    
  13     23     16    
0.8 12 16 4 17 15 2   1.2 1.0
  17     16          
  20     13          
4 19 18 4 8 13 4   1.3 0.8
  14     16          
  22     15          
20 20 20 2 12 13 1   1.4 0.8
  18     13          
  21     14          
100 21 20 1 8 17 8 22 1.4 1.0
  19     22     21    
  19     20     20    
500 11 13 3 12 14 2 15 0.9 0.9
  11     15     11    
  16     14     12    
 pos. control 2 -AA [10]       160 200 67     12.5
        162          
        277          
 pos. control MNNG [5] 1250 1260 10         90.0  
  1270                
  1260                

*: S9 -fraction/cofactors = 1:9

Table 4: Standard Plate Test, Strain TA 100

 

revertants / plate

titer dil.

quotient

dose [µg/plate] -S9 M SD +S9* M SD exp-6 -S9

+S9*

 neg. control DMSO 111 106 6 132 129 3 20 1.0 1.0
  109     127     11    
  99     129     35    
0.8 94 97 3 144 137 6   0.9 1.1
  100     133          
  97     133          
4 103 115 11 115 114 11   1.1 0.9
  124     125          
  119     103          
20 117 114 6 149 126 21   1.1 1.0
  107     120          
  118     109          
100 107 112 6 108 129 18 14 1.1 1.0
  109     135     18    
  119     143     12    
500 120 108 12 120 109 12 14 1.0  0.8
  97     97     10    
  107     110     10    
 pos. control 2 -AA [10]       1010 1105 85     8.5
        1130          
        1175          
 pos. control MNNG [5] 1340 1197 127         11.3  
  1150                
  1100                

*: S9 -fraction/cofactors = 1:9

Table 5: Standard Plate Test, Strain TA 1537

 

revertants / plate

titer dil.

quotient

dose [µg/plate] -S9 M SD +S9* M SD exp-6 -S9

+S9*

 neg. control DMSO 19 11 7 9 12 2 14 1.0 1.0
  7     13     18    
  8     13     19    
0.8 8 8 1 16 12 5   0.7 1.1
  9     14          
  7     7          
4 11 9 6 11 10 2   0.8 0.8
  14     10          
  2     8          
20 7 8 3 14 13 2   0.7 1.1
  5     10          
  11     14          
100 9 11 2 14 11 3 5 1.0 0.9
  13     10     3    
  11     8     7    
500 13 12 2 9 8 1 6 1.0  0.7
  9     8     5    
  13     8     8    
 pos. control 2 -AA [10]       300 265 34     22.7
        233          
        261          
 pos. control AAC [100] 374 464 95         40.9  
  455                
  563                

*: S9 -fraction/cofactors = 1:9

Table 6: Preincubation Test, Strain TA 1535

 

revertants / plate

titer dil.

quotient

 dose [µg/plate] -S9 M SD +S9* M SD exp-6 -S9

+S9*

 neg. control DMSO 15 17 3 13 15 2 14 1.0 1.0
  16     16     16    
  21     16     10    
0.8 21 21 2 22 21 6   1.2 1.4
  22     15          
  19     26          
4 27 24 4 17 17 1   1.4 1.1
  25     17          
  19     16          
20 26 25 1 18 18 2   1.4 1.3
  24     21          
  24     19          
100 22 21 4 14 16 4 5 1.2 1.1
  17     14     8    
  24     21     2    
500 B 0 0 B - - 0 0.0 -
  0     B     0    
  0     B     0    
 pos. control 2 -AA [10]       134 170 33     11.4
        198          
        179          
 pos. control MNNG [5] 851 814 49         47.0  
  832                
  759                

*: S9 -fraction/cofactors = 1:9;       B: reduced his- background

Table 7: Preincubation Test, Strain TA 100

 

revertants / plate

titer dil.

quotient

 dose [µg/plate] -S9 M SD +S9* M SD exp-6 -S9

+S9*

 neg. control DMSO 112 11 6 102 105 5 27 1.0 1.0
  116     111     19    
  104     102     23    
0.8 90 96 15 122 121 7   0.9 1.1
  84     113          
  113     127          
4 116 117 11 159 150 8   1.1 1.4
  107     143          
  128     149          
20 129 127 14 111 108 4   1.1 1.0
  112     110          
  139     103          
100 113 124 9 118 110 9 3 1.1 1.1
  129     113     8    
  129     100     2    
500 0 0 0 B - - 0 0.0 -
  0     B     0    
  0     B     0    
 pos. control 2 -AA [10]       848 1133 341     10.8
        1040          
        1510          
 pos. control MNNG [5] 684 675 10         6.1  
  676                
  664                

*: S9 -fraction/cofactors = 1:9;       B: reduced his- background

Table 8: Preincubation Test, Strain TA 1537

 

revertants / plate

titer dil.

quotient

 dose [µg/plate] -S9 M SD +S9* M SD exp-6 -S9

+S9*

 neg. control DMSO 11 9 2 11 11 1 3 1.0 1.0
  7     12     2    
  10     10     4    
0.8 14 12 4 5 10 5   1.3 0.9
  7     14          
  14     10          
4 7 8 2 11 11 2   0.9 1.0
  7     9          
  10     13          
20 10 8 2 10 10 3   0.8 0.9
  7     13          
  6     7          
100 13 12 3 12 9 3 4 1.3 0.8
  14     7     3    
  8     8     6    
500 0 0 0 B - - 0 0.0 -
  0     B     0    
  0     B     0    
 pos. control 2 -AA [10]       119 116 9     10.5
        123          
        105          
 pos. control AAC [100] 1200 1197 55         128.2  
  1250                
  1140                

*: S9 -fraction/cofactors = 1:9;       B: reduced his- background

Table 9: Preincubation Test, Strain TA 98

 

revertants / plate

titer dil.

quotient

 dose [µg/plate] -S9 M SD +S9* M SD exp-6 -S9

+S9*

 neg. control DMSO 30 31 1 35 35 5 21 1.0 1.0
  32     31     15    
  30     40     23    
0.8 17 20 4 41 35 6   0.6 1.0
  24     30          
  18     35          
4 21 23 3 40 38 4   0.7 1.1
  21     33          
  26     40          
20 20 22 7 34 36 3   0.7 1.0
  16     40          
  29     35          
100 14 20 6 30 31 3 0 0.7 0.9
  24     28     0    
  23     34     0    
500 0 0 0 B - - 0 0.0 -
  0     B     0    
  0     B     0    
 pos. control 2 -AA [10]       1370 1437 59     40.7
        1480          
        1460          
 pos. control NPD [10] 1320 1197 120         39.0  
  1190                
  1080                

*: S9 -fraction/cofactors = 1:9;       B: reduced his- background

Table 10: Standard Plate Test, Strain TA 98

 

revertants / plate

titer dil.

quotient

 dose [µg/plate] -S9 M SD +S9* M SD exp-6 -S9

+S9*

 neg. control DMSO 21 22 3 39 38 4 21 1.0 1.0
  25     41     15    
  19     33     29    
0.8 19 24 5 37 39 2   1.1 1.0
  25     39          
  29     40          
4 26 27 1 31 33 2   1.2 0.9
  28     33          
  26     34          
20 25 28 7 34 31 3   1.3 0.8
  22     28          
  36     32          
100 25 26 1 28 29 2 11 1.2 0.8
  27     32     15    
  27     28     16    
500 24 24 5 28 28 0 14 1.1  0.7
  20     28     9    
  29     28     9    
 pos. control 2 -AA [10]       1260 1132 133     30.0
        995          
        1140          
 pos. control NPD [10] 825 794 35         36.6  
  801                
  756                

*: S9 -fraction/cofactors = 1:9

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative