Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31st December 1999 to 3rd January 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study reported to have been conducted in accordance with OECD Guideline 471 and EU Method B13/14, however the information on all strains except TA100 was apparently deleted, therefore the test only has limited ability to detect mutations induced by base-pair substitution. However, the study was performed and reported to a good standard. Therefore it cannot be confirmed whether the test substance is mutagenic or not based on the results of the current study. No information on GLP status of the study provided.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(only TA100 used)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
(only TA100 tested)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
(R)-2,2,3-trimethylcyclopent-3-ene-1-acetaldehyde
EC Number:
224-815-8
EC Name:
(R)-2,2,3-trimethylcyclopent-3-ene-1-acetaldehyde
Cas Number:
4501-58-0
Molecular formula:
C10H16O
IUPAC Name:
(R)-2,2,3-trimethylcyclopent-3-ene-1-acetaldehyde
Details on test material:
- Name of test material (as cited in study report): Campholen aldehyde
- Storage condition of test material: cool and dry

Method

Target gene:
Histidine operon hisG46
Species / strain
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: 1.5 % agar in Vogel-Bonner medium E with 2 % glucose
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
15, 50, 150, 500, 1500 and 5000 µg per plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene and sodium azide both administered at 0.7 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72h

NUMBER OF REPLICATIONS: Three per test concentration
Evaluation criteria:
Plates were counted for number of revertant colonies (his+ revertants) and examined for the existence of a normal background lawn and/or precipitates and microscopically for microcolony growth.

When there was any doubt about the nature of the colonies scored as revertants, and when positive mutagenic results are obtained, the genotype of the revertant colonies are spot-checked streaking on histidine free plates.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
slightly bacteriotoxic at 1500 µg/plate; background lawn was nearly absent at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Induction of revertants in S. typhimurium by Campholen aldehyde in the absence of a metabolising system

 Substance  Concentration (µg/plate)  S9  Number of revertants per plate TA100      
       Counts  Mean  SD
 Control  0  -  134    
       162  154  16
       167    
 Solvent control  0  -  143    
       151  157  15
       176    
 Campholen aldehyde  15  -  162    
       142  157  11
       166    
 Campholen aldehyde  50  -  140    
       141  136  7
       127    
 Campholen aldehyde  150  -  165    
       144  152  10
       146    
 Campholen aldehyde  500  -  119    
       129  134  16
       154    
 Campholen aldeyhde  1500  -  64    
       112  82 T  22
     71    
 Campholen aldehyde  5000  -  0    
       0  0 T  0
       0    
 NaN3  0.7  -  432    
       446  434  10
       423    

T - bacteriotoxic

Table 2: Induction of revertants inS. typhimuriumby Campholen aldehyde in the presence of a metabolising system

Substance   Concentration (µg/plate)  S9  Number of revertants per plate TA100      
       Counts  Mean  SD
 Control  0  +  149    
       171  148  21
       123    
 Solvent control  0  +  163    
       127  150  18
       161    
 Campholen aldehyde  15  +  141    
       156  152  9
       158    
 Campholen aldehyde  50  +  159    
       190  166  19
       148    
 Campholen aldehyde  150  +  170    
       151  160  9
       160    
 Campholen aldehyde  500  +  124    
       106  122  14
       137    
 Campholen aldehyde  1500  +  134    
       119  126 T  7
       125    
 Campholen aldehyde  5000  +  0    
       0  0 T  0
       0    
 2 -AA  0.7  +  1356    
       1464  1330  122
       1170    

T - bacteriotoxic

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation in the S. typhimurium strain TA100

Under the conditions of the test, the test substance failed to induce a significant or dose-related increase in the mutation frequency of the strain TA100 in the absence and presence of metabolic activation. The estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dose level, using a X2-test, did not reveal a significant effect at any of the test points.
Executive summary:

The mutagenicity of the substance Campholen aldehyde was studied with the mutant strain TA100 of Salmonella typhimurium. The investigations were carried using the standard plate incorporation assay with and without liver homogenates (S9) from Aroclor 1254 pretreated male rats as the metabolic activation system.

Campholen aldehyde was dissolved in DMSO and tested in concentrations of 15 to 5000 µg per plate in the presence and absence of S9. In the absence and presence of S9-mix, Campholen aldehyde was slightly bacteriotoxic towards the strain TA100 at 1500 µg/plate and at 5000 µg/plate background lawn was nearly absent and no revertants were found.

Sodium azide and 2 -aminoanthracene served as positive controls to confirm the reversion properties and the specificity of the bacterial strain as well as the efficacy of the metabolising system.

In the concentration range investigated, Campholen aldehyde did not induce any increase in the mutation frequency of the tester strain TA100 in the presence and absence of a metabolic activation system.

In conclusion, these results indicate that Campholen aldehyde, under the experimental conditions described, was not mutagenic to Salmonella typhimurium strain TA100 in the presence and absence of a metabolising system.