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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro:

These endpoints were fulfilled using read across from Citonellol (3,7-dimethyloct-6-en-1-ol), Nerol (cis-3,7-dimethyl-2,6-octadien-1-ol ) and Geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol). Data from tests performed with the structurally comparable compounds nerol (cis-3,7-dimethyl-2,6-octadien-1-ol) or the respective stereoisomer geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol) were used to address this endpoint via read across. Inclusion of nerol/geraniol into this assessment is justified by the structural similarity with citronellol (3,7-dimethyloct-6-en-1-ol). These structures are identical except for an additional double bond found in nerol/geraniol. The structure of nerol/geraniol is considered to represent a worst case when compared to citronellol based on this second double bond as possible additional reactive feature. A putative steric hindrance to the action of cytochromes such as those present in the S9 mix due to the additional double bond is not to be expected. Furthermore, relevant physicochemical parameters show comparability between nerol/geraniol and citronellol (molecular weight of 154.2 and 156.3 ; log Pow at 2.6 and 3.41;  vapour pressure of 1 and 8.6 Pa; water solubility of 769 and 307 mg/l respectively).

Mutagenicity of citronellol in bacteria was analyzed in a study performed under GLP according to OECD guideline 471 and EU method B.13/14 (BASF, 1991). In this study bacteria strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 were treated with citronellol at concentrations of 8 - 500 µg/plate with and without metabolic activation by S9 fraction from Aroclor induced Sprague-Dawley rats. The strains S. typhimurium TA 100 and TA 98 were also exposed to 5000 µg/plate citronellol. Although bacteriotoxic effects at doses >= 500 µg/plate were noted, no increase in revertant colonies was observed. Therefore citronellol was not mutagenic in bacteria under the chosen testing conditions.

In a study in literature, performed by the National Toxicology Program (NTP) of the US National Institute of Health according to internal guidelines, bacteria strains S. typhimurium TA 1535, TA 97, TA 98 and TA 100 were treated with concentrations of 0, 1, 3, 10, 33, 100, 333 mg/plate citronellol in DMSO with and without metabolic activation by induced male Sprague Dawley rat liver S9 mix and induced male Syrian hamster liver S9 mix (NTP, 2002). Although bacteriotoxic effects were seen in the highest dose experiments, no increase in revertant colonies was observed. Therefore citronellol was not mutagenic in bacteria under the chosen testing conditions.

In order to support the data available for citronellal, data from an AMES test performed with the structurally comparable compound nerol (cis-3,7-dimethyl-2,6-octadien-1-ol) are included into the weight of evidence.

Genetic toxicity of nerol was analyzed in a bacterial reverse mutation assay performed under GLP according to OECD guideline 471 (Symrise, 2000). Bacteria strains S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were treated with nerol at concentrations of 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation by Aroclor-induced rat liver S9 mix. As a result, cytotoxicity was observed at 1500 ug/plate in TA100 and TA 102, and in TA98, TA1535 and TA1537 at a concentration of 5000 µg/plate. However, nerol was found to be not mutagenic under conditions of the study.

Overall in a weight of evidence and on the basis of the data available, citronellol is considered to be not mutagenic in bacteria.

 

In a chromosomal aberration test, concentrations of 0.0313, 0.0625, 0.125 mg/ml geraniol were tested in mammalian CHL cells 1-164 cells for clastogenic effects (Ishidate, 1984). After 48 h, 8% of the cells were polyploid (slightly, but significant elevated compared to control), and 4% of the cells showed chromosomal aberrations (not elevated compared to control). Thus, the test results were regarded as ambiguous.

In the other study, mammalian cells concentrations of 0, 33.3, 100, 333 and 1000 µM were tested in CHO cells during a sister chromatid exchange assay (Sasaki, 1989). Although cytotoxicity was noted at a concentration of 1000 µM, no substance induced chromatid exchange was detected.

Link to relevant study records

Referenceopen allclose all

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
Reason / purpose:
reference to same study
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:
negative
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: Comparable to guideline study with acceptable restrictions
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: NTP standard protocol
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Mortelmans K, Zeiger E. The Ames Salmonella/microsome mutagenicity assay. Mutat Res. 2000 Nov 20;455(1-2):29-60.

GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
His operon
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Metabolic activation system:
induced male Sprague Dawley rat liver S9 and induced male Syrian hamster liver S9
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Metabolic activation system:
induced male Sprague Dawley rat liver S9 and induced male Syrian hamster liver S9
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Metabolic activation system:
induced male Sprague Dawley rat liver S9 and induced male Syrian hamster liver S9
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Metabolic activation system:
induced male Sprague Dawley rat liver S9 and induced male Syrian hamster liver S9
Test concentrations with justification for top dose:
0, 1, 3, 10, 33, 100, 333 µg/ plate
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
Negative controls:
not specified
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene; TA 97, TA 98, TA 100, TA 1535
Remarks:
with metabolic activation
Negative controls:
not specified
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without metabolic activation Migrated to IUCLID6: TA 98
Negative controls:
not specified
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation Migrated to IUCLID6: TA100 and TA1535
Negative controls:
not specified
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation Migrated to IUCLID6: TA 97
Details on test system and conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: usually 2 days
Evaluation criteria:
Pattern and the strength of the mutant response are taken into account in determining the mutagenicity of a chemical. All observed responses are verified in repeat tests. If no increase in mutant colonies is seen after testing several strains under several different culture conditions, the test chemical is considered to be nonmutagenic in the Ames test.
Species / strain:
other: TA 97, TA 98, TA 100, TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
at 333 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: observed at 333 mg/plate
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: acceptable, well documented publication
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Composition 0
Composition 0
Composition 0
Qualifier:
no guideline followed
Principles of method if other than guideline:
chromosomal abberration tests in vitro using a Chinese hamster fibroblast cell line
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Test material information:
Composition 1
Species / strain:
other: CHL cells 1-164
Metabolic activation:
without
Test concentrations with justification for top dose:
0.0313, 0.0625, 0.125 mg/ml
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
Details on test system and conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 24 h or 48 h
- Fixation time (start of exposure up to fixation or harvest of cells): 2 h


SPINDLE INHIBITOR (cytogenetic assays): colcemid, 0.2 µg/ml
STAIN (for cytogenetic assays): Giemsa


NUMBER OF CELLS EVALUATED: 100
Evaluation criteria:
- The results were considered negative if the incidence of aberrations was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%.
- When dose-response relationships were not found, additional experiments were carried out at similar dose levels.
Statistics:
- For a quantitative evaluation of the clastogenic potential of positive samples, the D20 (dose in mg/ml at which structural aberrations (including gaps) were detected in 20% of metaphases observed) was determined.
- Additionally, the TR value (indicates frequency of cells with exchange-type aberrations per unit dose (mg/ml)) was calculated.
- A high TR value exists for chemicals that show carcinogenic potential in animals.
Species / strain:
other: CHL cells 1-164
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
24 hour-Treatment:

Percentage of polyploid cells (%):
solvent 0
0.0313 mg/mL 2
0.0625 mg/mL 6
0.125 mg/mL 4

Cells with structural chromosome aberrations (%) (total):
solvent 0
0.0313 mg/mL 1
0.0625 mg/mL 2
0.125 mg/mL 3


48 hour-Treatment:

Percentage of polyploid cells (%):
solvent 0
0.0313 mg/mL 0
0.0625 mg/mL 4
0.125 mg/mL 8

Cells with structural chromosome aberrations (%) (total):
solvent 1
0.0313 mg/mL 0
0.0625 mg/mL 2
0.125 mg/mL 4
Conclusions:
Interpretation of results:
ambiguous
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: acceptable, well documented publication
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Composition 0
Qualifier:
no guideline followed
Principles of method if other than guideline:
in vitro cytogenetic assay in Chinese hamster ovary cells (CHO-K1)
GLP compliance:
not specified
Type of assay:
sister chromatid exchange assay in mammalian cells
Test material information:
Composition 1
Species / strain:
other: Chinese hamster CHO-K1 cells
Metabolic activation:
without
Test concentrations with justification for top dose:
0; 33,3; 100; 333; 1000 µM
Details on test system and conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 21 h
- Selection time (if incubation with a selection agent): 21 h


SELECTION AGENT (mutation assays): mitomycin C
SPINDLE INHIBITOR (cytogenetic assays): colchicine, 50 g/ml, 2 h
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: 3


NUMBER OF CELLS EVALUATED: 50 metaphases /culture
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
other: at 1000 µM
Conclusions:
Interpretation of results:
negative
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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study according to GLP
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
no preincubation method performed
GLP compliance:
yes
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 liver homogenate from Aroclor 1254 pretreated male rats
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenate from Aroclor 1254 pretreated male rats
Test concentrations with justification for top dose:
- Prescreen: 5 - 5000 µg/plate
- Test: 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
Negative controls:
yes
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without metabolic activation Migrated to IUCLID6: 2.5 µg/plate (TA98) in DMSO
Negative controls:
yes
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation Migrated to IUCLID6: 0.7 µg/plate (TA100, TA1535) in water
Negative controls:
yes
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation Migrated to IUCLID6: 50 µg/plate (TA1537) in water
Negative controls:
yes
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation Migrated to IUCLID6: 0.15 µg/plate (TA102) in water
Negative controls:
yes
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 0.8 µg/plate (TA98, TA100, TA102, TA1535), 1.7 µg/plate (TA1537) in DMSO
Remarks:
with metabolic activation
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 - 72 h, 37°C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduction in revertant colonies and/or diminution of background lawn
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test compound an the plates was not observed.

COMPARISON WITH HISTORICAL CONTROL DATA:
The number of spontaneous revertants observed using each of the five strains was close to these previously established in our laboratory and was within the range obtained by Ames et al. (1975) as well as reported by De Serres and Shelby (1979). Similarly, the results with the positive control substances confirmed the known reversion properties and specificity of the tester strains as well as the full activity of the metabolizing system.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Without S9-mix: NEROL SUPRA was bacteriotoxic towards all strains at 1500 µg/plate.
WithS9-mix: NEROL SUPRA was bacteriotoxic towards the strains TA100 and TA102 at 1500 µg/plate and towards the strains TA98, TA1535, and TA1537 at 5000 µg/plate.
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
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: Comparable to guideline study with acceptable restrictions
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:
other: NTP standard protocol
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Mortelmans K, Zeiger E. The Ames Salmonella/microsome mutagenicity assay. Mutat Res. 2000 Nov 20;455(1-2):29-60.

GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
His operon
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Metabolic activation system:
induced male Sprague Dawley rat liver S9 and induced male Syrian hamster liver S9
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Metabolic activation system:
induced male Sprague Dawley rat liver S9 and induced male Syrian hamster liver S9
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Metabolic activation system:
induced male Sprague Dawley rat liver S9 and induced male Syrian hamster liver S9
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Metabolic activation system:
induced male Sprague Dawley rat liver S9 and induced male Syrian hamster liver S9
Test concentrations with justification for top dose:
0, 1, 3, 10, 33, 100, 333 µg/ plate
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
Negative controls:
not specified
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene; TA 97, TA 98, TA 100, TA 1535
Remarks:
with metabolic activation
Negative controls:
not specified
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without metabolic activation Migrated to IUCLID6: TA 98
Negative controls:
not specified
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation Migrated to IUCLID6: TA100 and TA1535
Negative controls:
not specified
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation Migrated to IUCLID6: TA 97
Details on test system and conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: usually 2 days
Evaluation criteria:
Pattern and the strength of the mutant response are taken into account in determining the mutagenicity of a chemical. All observed responses are verified in repeat tests. If no increase in mutant colonies is seen after testing several strains under several different culture conditions, the test chemical is considered to be nonmutagenic in the Ames test.
Species / strain:
other: TA 97, TA 98, TA 100, TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
at 333 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: observed at 333 mg/plate
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
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: acceptable, well documented publication
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:
reference to same study
Reason / purpose:
read-across source
Related information:
Composition 1
Reference:
Composition 0
Composition 0
Composition 0
Composition 0
Qualifier:
no guideline followed
Principles of method if other than guideline:
chromosomal abberration tests in vitro using a Chinese hamster fibroblast cell line
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Test material information:
Composition 1
Species / strain:
other: CHL cells 1-164
Metabolic activation:
without
Test concentrations with justification for top dose:
0.0313, 0.0625, 0.125 mg/ml
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
Details on test system and conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 24 h or 48 h
- Fixation time (start of exposure up to fixation or harvest of cells): 2 h


SPINDLE INHIBITOR (cytogenetic assays): colcemid, 0.2 µg/ml
STAIN (for cytogenetic assays): Giemsa


NUMBER OF CELLS EVALUATED: 100
Evaluation criteria:
- The results were considered negative if the incidence of aberrations was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%.
- When dose-response relationships were not found, additional experiments were carried out at similar dose levels.
Statistics:
- For a quantitative evaluation of the clastogenic potential of positive samples, the D20 (dose in mg/ml at which structural aberrations (including gaps) were detected in 20% of metaphases observed) was determined.
- Additionally, the TR value (indicates frequency of cells with exchange-type aberrations per unit dose (mg/ml)) was calculated.
- A high TR value exists for chemicals that show carcinogenic potential in animals.
Species / strain:
other: CHL cells 1-164
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
24 hour-Treatment:

Percentage of polyploid cells (%):
solvent 0
0.0313 mg/mL 2
0.0625 mg/mL 6
0.125 mg/mL 4

Cells with structural chromosome aberrations (%) (total):
solvent 0
0.0313 mg/mL 1
0.0625 mg/mL 2
0.125 mg/mL 3


48 hour-Treatment:

Percentage of polyploid cells (%):
solvent 0
0.0313 mg/mL 0
0.0625 mg/mL 4
0.125 mg/mL 8

Cells with structural chromosome aberrations (%) (total):
solvent 1
0.0313 mg/mL 0
0.0625 mg/mL 2
0.125 mg/mL 4
Conclusions:
Interpretation of results (migrated information):
ambiguous
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
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: acceptable, well documented publication
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:
reference to same study
Reason / purpose:
read-across source
Related information:
Composition 1
Reference:
Composition 0
Composition 0
Qualifier:
no guideline followed
Principles of method if other than guideline:
in vitro cytogenetic assay in Chinese hamster ovary cells (CHO-K1)
GLP compliance:
not specified
Type of assay:
sister chromatid exchange assay in mammalian cells
Test material information:
Composition 1
Species / strain:
other: Chinese hamster CHO-K1 cells
Metabolic activation:
without
Test concentrations with justification for top dose:
0; 33,3; 100; 333; 1000 µM
Details on test system and conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 21 h
- Selection time (if incubation with a selection agent): 21 h


SELECTION AGENT (mutation assays): mitomycin C
SPINDLE INHIBITOR (cytogenetic assays): colchicine, 50 g/ml, 2 h
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: 3


NUMBER OF CELLS EVALUATED: 50 metaphases /culture
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
other: at 1000 µM
Conclusions:
Interpretation of results (migrated information):
negative
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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study according to GLP
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:
no preincubation method performed
GLP compliance:
yes
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 liver homogenate from Aroclor 1254 pretreated male rats
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenate from Aroclor 1254 pretreated male rats
Test concentrations with justification for top dose:
- Prescreen: 5 - 5000 µg/plate
- Test: 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
Negative controls:
yes
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without metabolic activation Migrated to IUCLID6: 2.5 µg/plate (TA98) in DMSO
Negative controls:
yes
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation Migrated to IUCLID6: 0.7 µg/plate (TA100, TA1535) in water
Negative controls:
yes
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation Migrated to IUCLID6: 50 µg/plate (TA1537) in water
Negative controls:
yes
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation Migrated to IUCLID6: 0.15 µg/plate (TA102) in water
Negative controls:
yes
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 0.8 µg/plate (TA98, TA100, TA102, TA1535), 1.7 µg/plate (TA1537) in DMSO
Remarks:
with metabolic activation
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 - 72 h, 37°C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduction in revertant colonies and/or diminution of background lawn
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test compound an the plates was not observed.

COMPARISON WITH HISTORICAL CONTROL DATA:
The number of spontaneous revertants observed using each of the five strains was close to these previously established in our laboratory and was within the range obtained by Ames et al. (1975) as well as reported by De Serres and Shelby (1979). Similarly, the results with the positive control substances confirmed the known reversion properties and specificity of the tester strains as well as the full activity of the metabolizing system.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Without S9-mix: NEROL SUPRA was bacteriotoxic towards all strains at 1500 µg/plate.
WithS9-mix: NEROL SUPRA was bacteriotoxic towards the strains TA100 and TA102 at 1500 µg/plate and towards the strains TA98, TA1535, and TA1537 at 5000 µg/plate.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Genetic toxicity in vivo:

These endpoints were fulfilled using read across from a reaction mass of Nerol (cis-3,7-dimethyl-2,6-octadien-1-ol ) and Geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol). Data from tests performed with the structurally comparable compounds nerol (cis-3,7-dimethyl-2,6-octadien-1-ol) or the respective stereoisomer geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol) were used to address this endpoint via read across. Inclusion of nerol/geraniol into this assessment is justified by the structural similarity with citronellol (3,7-dimethyloct-6-en-1-ol). These structures are identical except for an additional double bond found in nerol/geraniol. The structure of nerol/geraniol is considered to represent a worst case when compared to citronellol based on this second double bond as possible additional reactive feature. A putative steric hindrance to the action of cytochromes such as those present in the S9 mix due to the additional double bond is not to be expected. Furthermore, relevant physicochemical parameters show comparability between nerol/geraniol and citronellol (molecular weight of 154.2 and 156.3 ; log Pow at 2.6 and 3.41;  vapour pressure of 1 and 8.6 Pa; water solubility of 769 and 307 mg/l respectively).

 

The reaction mass of geraniol and nerol was tested in a MNT in NMRI mice (BASF SE, 2010). For this purpose, the test substance, dissolved in DMSO and emulsified in corn oil, was administered once orally to male animals at dose levels of 375 mg/kg, 750 mg/kg and 1 500 mg/kg body weight in a volume of 10 mL/kg body weight in each case. The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 1 500 mg/kg body weight and in the vehicle controls. In the test groups of 750 mg/kg and 375 mg/kg body weight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2 000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2 000 polychromatic erythrocytes were also recorded. As vehicle control, male mice were administered merely the vehicle, DMSO/corn oil (ratio 2:3), by the same route and in the same volume as the animals of the dose groups, which gave frequencies of micronucleated polychromatic erythrocytes within the historical vehicle control data range. Both positive control substances, cyclophosphamide for clastogenicity and vincristine sulfate for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected.

According to the results of the study, the single oral administration of the reaction mass of geraniol and nerol did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was within the range of the concurrent vehicle control in all dose groups and at all sacrifice intervals and within the range of the historical vehicle control data. Thus, under the experimental conditions of this study, the reaction mass of geraniol and nerol does not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo.

Overall in a weight of evidence, the data of the structural analogue provide evidence for an absence of any cytogenic activity of citronellol.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study according to OECD guideline and GLP
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Test material information:
Composition 1
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Age at study initiation: 5-8 weeks
- Weight at study initiation: 28.15 g
- Assigned to test groups randomly: yes, according to a randomization plan prepared with an appropriate computer program
- Housing: single in Makrolon cages, type M II
- Diet (e.g. ad libitum): standardized pellet feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): drinking water from bottles, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): fully air conditioned rooms with central air conditioning
- Photoperiod (hrs dark / hrs light): 12:12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO/corn oil (ratio 2:3)
- Justification for choice of solvent/vehicle: limited solubility of test substance in water and better volume for administration
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw
Duration of treatment / exposure:
once
Frequency of treatment:
once
Post exposure period:
24 and 48 hours
Remarks:
Doses / Concentrations:
375 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
750 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1500 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
- cyclophosphamide (CPP): 20 mg/kg bw
- vincristine sulfate (VCR): 0.15 mg/kg bw
Tissues and cell types examined:
- Clinical examinations: After treatment up to the time of sacrifice, the animals were examined for any clinically evident signs of toxicity several times.
- Preparation of the bone marrow: The bone marrow was prepared according to the method described by Schmid and Salamone et al.
- MICROSCOPIC EVALUATION
In general, 2 000 polychromatic erythrocytes (PCE) were evaluated for the occurrence of micronuclei from each animal of every test group, so in total 10 000 PCEs were scored per test group. The normochromatic erythrocytes (= normocytes / NCE) were also scored. The following parameters were recorded:
• Number of polychromatic erythrocytes
• Number of polychromatic erythrocytes containing micronuclei
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the vehicle control group provides an index of a chromosome-breaking (clastogenic) effect or damage of the mitotic apparatus (aneugenic activity) of the test substance administered.
• Number of normochromatic erythrocytes
• Number of normochromatic erythrocytes containing micronuclei
The number of micronuclei in normochromatic erythrocytes at the early sacrifice interval shows the situation before test substance administration and may serve as a control value. A test substance induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice interval.
• Ratio of polychromatic to normochromatic erythrocytes
An alteration of this ratio indicates that the test substance actually reached the bone marrow, means the target determined for genotoxic effects.
• Number of small micronuclei (d < D/4) and of large micronuclei (d ≥ D/4)
[d = diameter of micronucleus, D = cell diameter]
The size of micronuclei may indicate the possible mode of action of the test substance, i.e. a clastogenic effect (d < D/4) or a spindle poison effect (d ≥ D/4).
Slides were coded before microscopic analysis.
Details of tissue and slide preparation:
Preparation of the bone marrow
- The animals were anesthetized with isoflurane and afterwards sacrificed by cervical dislocation. Then the two femora were prepared by dissection and removing all soft tissues.
- After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum (FCS) which was preheated up to 37°C (about 2 mL/femur).
- The suspension was mixed thoroughly with a pipette and centrifuged at 300 x g for 5 minutes. The supernatant was removed and the precipitate was resuspended in about 50 μL fresh FCS.
- One drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges. The preparations were dried in the air and subsequently stained.

Staining of the slides
- The slides were stained with eosin and methylene blue (modified May-Grünwald solution or Wrights solution) for about 5 minutes.
- After briefly rinsing in purified water, the preparations were soaked in purified water for about 2 - 3 minutes.
- Subsequently, the slides were stained with Giemsa solution (15 mL Giemsa plus 185 mL purified water) for about 15 minutes.
- After rinsing twice in purified water and clarifying in xylene, the preparations were mounted in Corbit-Balsam.
Evaluation criteria:
Acceptance criteria
The mouse micronucleus test is considered valid if the following criteria are met:
• The quality of the slides must allow the evaluation of a sufficient number of analyzable
cells; i. e. ≥ 2 000 PCEs per animal and a clear differentiation between PCEs and NCEs.
• The ratio of PCEs/NCEs in the concurrent vehicle control animals has to be within the normal range for the animal strain selected.
• The number of cells containing micronuclei in vehicle control animals has to be within the range of the historical vehicle control data both for PCEs and for NCEs.
• The two positive control substances have to induce a distinct increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.

Assessment criteria
A finding is considered positive if the following criteria are met:
• Statistically significant and dose-related increase in the number of PCEs containing micronuclei.
• The number of PCEs containing micronuclei has to exceed both the concurrent vehicle control value and the range of the historical vehicle control data.
A test substance is considered negative if the following criteria are met:
• The number of cells containing micronuclei in the dose groups is not statistically significant increased above the concurrent vehicle control value and is within the range of the historical vehicle control data.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN (BASF SE). The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there are statistically significant differences between the untreated control group and the treated dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal were used as a criterion for the rank determination for the U test. Statistical significances were identified as follows:
* p ≤ 0.05
** p ≤ 0.01
However, both biological relevance and statistical significance were considered together.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
piloerection and hunched posture in high dose
Vehicle controls valid:
yes
Positive controls valid:
yes
Additional information on results:
According to the results of the present study, there are no statistical significances or biologically relevant differences in the frequency of erythrocytes containing micronuclei either between the vehicle control groups and the three dose groups (375 mg/kg, 750 mg/kg and 1 500 mg/kg) or between the two sacrifice intervals (24 and 48 hours). The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d ≥ D/4) did not deviate from the vehicle control values at any of the sacrifice intervals and was within the historical vehicle control data range.
In this study, after single oral administration of the vehicle DMSO/corn oil the ratio of PCEs/NCEs in the vehicle control animals at both sacrifice intervals was within the normal range for the animal strain selected. Besides the number of cells containing micronuclei in these vehicle control animals was within the range of the historical vehicle control data both for PCEs and for NCEs.
In addition, both positive control substances, cyclophosphamide and vincristine sulfate, induced a statistically significant increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.

Summary table – Induction of Micronuclei in bone marrow cells

Test group

Sacrificeinterval

[hrs]

AnimalNo.

Micronuclei in PCE

Numberof NCEc

totala[‰]

largeb[‰]

Vehicle control

DMSO/corn oil

24

5

2.4

0.1

3 505 

Test substance

375 mg/kg bw.

24

5

1.3

0.0

3 490

Test substance

750 mg/kg bw.

24

5

1.3

0.0

3188

Test substance

1 500 mg/kg bw.

24

5

1.4

0.0

3 699

Positive control

cyclophosphamide

20 mg/kg bw.

24

5

13.7**

0.1

4 076

Positive control

vincristine sulfate

0.15 mg/kg bw.

24

5

47.9**

14.7**

5 879

Vehicle control

DMSO/corn oil

48

5

1.3

0.0

4 630

Test substance

1 500 mg/kg bw.

48

5

1.3

0.0

4 078

 

PCE = polychromatic erythrocytes

NCE = normochromatic erythrocytes

bw. = body weight

 

a= sum of small and large micronuclei

b= large micronuclei (indication for spindle poison effect)

c= number of NCEs observed when scoring 10 000 PCEs

 

* = p ≤ 0.05

** = p ≤ 0.01

Conclusions:
Interpretation of results: negative
According to the authors, under the experimental conditions chosen here, the test substance has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells of NMRI mice in vivo.
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: study according to OECD guideline and GLP
Reason / purpose:
read-across source
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Test material information:
Composition 1
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Age at study initiation: 5-8 weeks
- Weight at study initiation: 28.15 g
- Assigned to test groups randomly: yes, according to a randomization plan prepared with an appropriate computer program
- Housing: single in Makrolon cages, type M II
- Diet (e.g. ad libitum): standardized pellet feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): drinking water from bottles, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): fully air conditioned rooms with central air conditioning
- Photoperiod (hrs dark / hrs light): 12:12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO/corn oil (ratio 2:3)
- Justification for choice of solvent/vehicle: limited solubility of test substance in water and better volume for administration
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw
Duration of treatment / exposure:
once
Frequency of treatment:
once
Post exposure period:
24 and 48 hours
Remarks:
Doses / Concentrations:
375 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
750 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1500 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
- cyclophosphamide (CPP): 20 mg/kg bw
- vincristine sulfate (VCR): 0.15 mg/kg bw
Tissues and cell types examined:
- Clinical examinations: After treatment up to the time of sacrifice, the animals were examined for any clinically evident signs of toxicity several times.
- Preparation of the bone marrow: The bone marrow was prepared according to the method described by Schmid and Salamone et al.
- MICROSCOPIC EVALUATION
In general, 2 000 polychromatic erythrocytes (PCE) were evaluated for the occurrence of micronuclei from each animal of every test group, so in total 10 000 PCEs were scored per test group. The normochromatic erythrocytes (= normocytes / NCE) were also scored. The following parameters were recorded:
• Number of polychromatic erythrocytes
• Number of polychromatic erythrocytes containing micronuclei
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the vehicle control group provides an index of a chromosome-breaking (clastogenic) effect or damage of the mitotic apparatus (aneugenic activity) of the test substance administered.
• Number of normochromatic erythrocytes
• Number of normochromatic erythrocytes containing micronuclei
The number of micronuclei in normochromatic erythrocytes at the early sacrifice interval shows the situation before test substance administration and may serve as a control value. A test substance induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice interval.
• Ratio of polychromatic to normochromatic erythrocytes
An alteration of this ratio indicates that the test substance actually reached the bone marrow, means the target determined for genotoxic effects.
• Number of small micronuclei (d < D/4) and of large micronuclei (d ≥ D/4)
[d = diameter of micronucleus, D = cell diameter]
The size of micronuclei may indicate the possible mode of action of the test substance, i.e. a clastogenic effect (d < D/4) or a spindle poison effect (d ≥ D/4).
Slides were coded before microscopic analysis.
Details of tissue and slide preparation:
Preparation of the bone marrow
- The animals were anesthetized with isoflurane and afterwards sacrificed by cervical dislocation. Then the two femora were prepared by dissection and removing all soft tissues.
- After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum (FCS) which was preheated up to 37°C (about 2 mL/femur).
- The suspension was mixed thoroughly with a pipette and centrifuged at 300 x g for 5 minutes. The supernatant was removed and the precipitate was resuspended in about 50 μL fresh FCS.
- One drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges. The preparations were dried in the air and subsequently stained.

Staining of the slides
- The slides were stained with eosin and methylene blue (modified May-Grünwald solution or Wrights solution) for about 5 minutes.
- After briefly rinsing in purified water, the preparations were soaked in purified water for about 2 - 3 minutes.
- Subsequently, the slides were stained with Giemsa solution (15 mL Giemsa plus 185 mL purified water) for about 15 minutes.
- After rinsing twice in purified water and clarifying in xylene, the preparations were mounted in Corbit-Balsam.
Evaluation criteria:
Acceptance criteria
The mouse micronucleus test is considered valid if the following criteria are met:
• The quality of the slides must allow the evaluation of a sufficient number of analyzable
cells; i. e. ≥ 2 000 PCEs per animal and a clear differentiation between PCEs and NCEs.
• The ratio of PCEs/NCEs in the concurrent vehicle control animals has to be within the normal range for the animal strain selected.
• The number of cells containing micronuclei in vehicle control animals has to be within the range of the historical vehicle control data both for PCEs and for NCEs.
• The two positive control substances have to induce a distinct increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.

Assessment criteria
A finding is considered positive if the following criteria are met:
• Statistically significant and dose-related increase in the number of PCEs containing micronuclei.
• The number of PCEs containing micronuclei has to exceed both the concurrent vehicle control value and the range of the historical vehicle control data.
A test substance is considered negative if the following criteria are met:
• The number of cells containing micronuclei in the dose groups is not statistically significant increased above the concurrent vehicle control value and is within the range of the historical vehicle control data.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN (BASF SE). The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there are statistically significant differences between the untreated control group and the treated dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal were used as a criterion for the rank determination for the U test. Statistical significances were identified as follows:
* p ≤ 0.05
** p ≤ 0.01
However, both biological relevance and statistical significance were considered together.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
piloerection and hunched posture in high dose
Vehicle controls valid:
yes
Positive controls valid:
yes
Additional information on results:
According to the results of the present study, there are no statistical significances or biologically relevant differences in the frequency of erythrocytes containing micronuclei either between the vehicle control groups and the three dose groups (375 mg/kg, 750 mg/kg and 1 500 mg/kg) or between the two sacrifice intervals (24 and 48 hours). The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d ≥ D/4) did not deviate from the vehicle control values at any of the sacrifice intervals and was within the historical vehicle control data range.
In this study, after single oral administration of the vehicle DMSO/corn oil the ratio of PCEs/NCEs in the vehicle control animals at both sacrifice intervals was within the normal range for the animal strain selected. Besides the number of cells containing micronuclei in these vehicle control animals was within the range of the historical vehicle control data both for PCEs and for NCEs.
In addition, both positive control substances, cyclophosphamide and vincristine sulfate, induced a statistically significant increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.

Summary table – Induction of Micronuclei in bone marrow cells

Test group

Sacrificeinterval

[hrs]

AnimalNo.

Micronuclei in PCE

Numberof NCEc

totala[‰]

largeb[‰]

Vehicle control

DMSO/corn oil

24

5

2.4

0.1

3 505 

Test substance

375 mg/kg bw.

24

5

1.3

0.0

3 490

Test substance

750 mg/kg bw.

24

5

1.3

0.0

3188

Test substance

1 500 mg/kg bw.

24

5

1.4

0.0

3 699

Positive control

cyclophosphamide

20 mg/kg bw.

24

5

13.7**

0.1

4 076

Positive control

vincristine sulfate

0.15 mg/kg bw.

24

5

47.9**

14.7**

5 879

Vehicle control

DMSO/corn oil

48

5

1.3

0.0

4 630

Test substance

1 500 mg/kg bw.

48

5

1.3

0.0

4 078

 

PCE = polychromatic erythrocytes

NCE = normochromatic erythrocytes

bw. = body weight

 

a= sum of small and large micronuclei

b= large micronuclei (indication for spindle poison effect)

c= number of NCEs observed when scoring 10 000 PCEs

 

* = p ≤ 0.05

** = p ≤ 0.01

Conclusions:
Interpretation of results (migrated information): negative
According to the aouthors, under the experimental conditions chosen here, the test substance has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells of NMRI mice in vivo.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

Based on negative results in in-vitro and in-vivo studies conducted, the substance does not need to be classified for germ cell mutagenicity.