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:
2006-04-11 to 2006-05-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to guideline
Guideline:
other: EEC Council Directive 2000/32 Annex 4D
Qualifier:
according to guideline
Guideline:
other: ICH S2A Genotoxicity:Specific Aspects of Regulatory Tests, Step5
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4'-Methylenebis(N-butoxycarbonylcyclohexanamine)
EC Number:
619-422-7
Cas Number:
99305-42-7
Molecular formula:
C23 H42 N2 O4
IUPAC Name:
4,4'-Methylenebis(N-butoxycarbonylcyclohexanamine)
Details on test material:
4,4'-Methylenebis(N-butoxycarbonylcyclohexanamine) of Degussa AG, batch 03.10.05/B-6320, purity 89.4 %

Method

Target gene:
Salmonella typhimurium strains: mutated his genes (his auxotroph)
Escherichia coli strain: mutated trp gene (trp auxotroph)
Species / strain
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100; E. coli WP2 uvrA
Additional strain / cell type characteristics:
other: Salmonella typhimurium strains: his auxotroph; Escherichia coli strain: trp auxotroph
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital / betanaphthoflavone  co-induced rat liver S9 fraction, lot 1934 from Moltox Molecular  Toxicology, Inc., prepared from male Sprague-Dawley rats
Test concentrations with justification for top dose:
Main Assay I (incorporation method): 5000; 2500; 1250; 625; 313 µg/plate (additional concentrations for S. typhimurium TA100 only: 156; 78.1 µg/plate) +/- S9 mix Main Assay II (Preincubation Method): 5000; 2500; 1250; 625; 313 µg/plate +/- S9 mix
Vehicle / solvent:
DMSO (dimethyl sulfoxide (CAS No.  67-68-5) )
Controls
Untreated negative controls:
yes
Remarks:
details see "Details on test system and conditions" below
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
details see "Details on test system and conditions" below
Positive control substance:
other: different positive control substances, see "Details on test system and conditions" below
Details on test system and experimental conditions:
Ames test
SYSTEM OF TESTING
- Species/cell type: Salmonella typhimurium TA 1535, TA 1537, TA 98, TA  100; Escherichia coli WP2 uvrA
- Metabolic activation system: Phenobarbital / betanaphthoflavone  co-induced rat liver S9 fraction, lot 1934 from Moltox Molecular  
Toxicology, Inc., prepared from male Sprague-Dawley rats
ADMINISTRATION: 
- Dosing:    
Preliminary toxicity test (1 replicate): 50; 158; 500; 1580; 5000  µg/plate (+/- metabolic activation)   
Plate incorporation test: 313; 625; 1250; 2500; 5000 µg/plate; TA 100  also 78.1 and 156 µg/plate (all +/- metabolic activation)   
Pre-incubation test: 313; 625; 1250; 2500; 5000 µg/plate (+/- metabolic  activation)
- Number of replicates: 3
- Application: Solution (100 mg/ml) in dimethyl sulfoxide (CAS No.  67-68-5)
- Positive and negative control groups and treatment:    
positive, TA 1535: 1 µg sodium azide/plate (- S9) / 1 µg  2-aminoanthracene/plate (+ S9)   
positive, TA 1537: 50 µg 9-aminoacridine/plate (- S9) / 1 µg  2-aminoanthracene/plate (+ S9)   
positive, WP2 uvrA: 500 µg Methylmethanesulfonate/plate (- S9) / 10 or  20 µg 2-aminoanthracene/plate (+ S9)   
positive, TA 98: 2 µg 2-nitrofluorene/plate (- S9) / 1 or 2 µg  2-aminoanthracene/plate (+ S9)   
positive, TA 100: 1 µg sodium azide/plate (- S9) / 1 or 2 µg  2-aminoanthracene/plate (+ S9)   
negative: untreated / solvent control (100 µl/plate) (pre-incubation:  50 µl/plate)   
sterility control; bacteria density control
- Pre-incubation: 30 minutes at 37 °C   
incubation approximately 72 hours at 37 °C
Evaluation criteria:
CRITERIA FOR EVALUATING RESULTS:    
ratio of mean revertant rates treated/control >= 2 at the highest dose level  or at two consecutive dose levels with generally positive dose-response  
relationship 
Statistics:
Regression analysis
Regression lines are calculated using a minimum of three lowest dose-levels, and then including the further dose-levels in turn. The correlation
co-efficient (r), the value of students "t" statistic, and the p-value for the regression lines are also given.

Results and discussion

Test results
Species / strain:
bacteria, other: Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100; Escherichia coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
With metabolic activation: 5000 µg/plate only in TA 100 strain (toxicity test);  Without metabolic activation: >= 1580 µg/plate only in TA 100 strain (toxicity test
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
GENOTOXIC EFFECTS: 
- With metabolic activation: none
- Without metabolic activation: none   The positive controls were functional.
PRECIPITATION CONCENTRATION: >= 2500 µg/plate (+/- metabolic  activation)
CYTOTOXIC CONCENTRATION:
- With metabolic activation: 5000 µg/plate only in TA 100 strain (toxicity test)
- Without metabolic activation: >= 1580 µg/plate only in TA 100 strain (toxicity  test)
Remarks on result:
other: strain/cell type: S. typhimurium TA 1535, TA 1537, TA 98, TA 100; E. coli WP2 uvrA
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

no further information

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that the test item H12MDU does not induce reverse mutation in Salmonella typhimurium or Escherichia coli under the reported
experimental conditions.
Executive summary:

The test item H12MDU was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium (TA 1535; TA1537, TA 98; TA100) and Escherichia coli (WP2 uvrA). The experiments were performed both in the absence and in the presence of metabolic activation using liver S9 mix from rats pre-treated with phenobarbitone and betanaphthoflavone.

The test item H12MDU did not induce two fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose-level, in any tester strain, in the absence or presence of S9 metabolism.

It is concluded that the test item H12MDU does not induce reverse mutation in Salmonella typhimurium or Escherichia coli under the reported experimental conditions.