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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999-01-12 to 1999-02-25-1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
yes
Remarks:
The study was performed only with strain TA98 and strain TA100
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1992
Deviations:
yes
Remarks:
The study was performed only with strain TA98 and strain TA100
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(3,5-Dihydroxyphenyl)-1-oxo-2-(N-1-(4-methoxyphenyl)isopropylamino)ethane Hydrobromide
EC Number:
928-779-0
Molecular formula:
C18-H21-N-O4 x HBr
IUPAC Name:
1-(3,5-Dihydroxyphenyl)-1-oxo-2-(N-1-(4-methoxyphenyl)isopropylamino)ethane Hydrobromide
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
No details on the source of the test material were provided. Batch number: 323.
- Expiration date of the lot/batch:
16 December 1999 (allocated by testing facility, 1 year after reciept of the test substance).
- Purity test date: No details reported.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark.
- Stability under test conditions: Stable The stability of the test substance in the vehicle was not indicated.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No details reported.
- Preliminary purification step (if any):No details reported.
- Final dilution of a dissolved solid, stock liquid or gel:No details reported.
- Final preparation of a solid: No details reported.

Method

Target gene:
Strain TA98 characterises the histidine mutation 'hisD3053/R-factor8' for frameshift mutations (* R-factor = plasmid pKM101 to increase error prone DNA repair).
Strain TA100 characterises the histidine mutation 'hisG46/R-factor' for base pair substitutions.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate (first experiment; TA98 and TA100 with and without metabolic activation)
500, 1000, 2000, 3000, 4000 and 5000 µg/plate (second experiment; TA100 with and without metabolic activation, TA98 without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (test substance, positive control substance methylmethanesulfonate, 2-aminoanthracene); Saline (positive control substance daunomycine)
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
other: 2-aminoanthracene and daunomycine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 each per concentration level and control

DETERMINATION OF CYTOTOXICITY
- Method: Bacterial background lawn, microcolonies
Evaluation criteria:
The assessment and interpretation of the results follows the OECD Guideline No. 471, i. e. a concentration-dependent increase in the number of revertants of at least one tester strain over the vehicle control value and/or outside the historical control range is indicative of genotoxic activity.

A test substance is considered negative (not mutagenic) in the test if: The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
A test substance is considered positive (mutagenic) in the test if: It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item did not precipitate in the top agar. Precipitation of the test item was not observed on the plates at the start or at the end of the incubation period in both tester strains.
COMPARISON WITH HISTORICAL CONTROL DATA: Yes

Any other information on results incl. tables

The negative and strain-specific positive control values were within the testing laboratory's background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Strain TA98

In the absence of S9 -mix, the test item showed negative responses over the entire dose range, i. e. no dose related, two-fold, increase in the number of revertants. In the presence of S9- mix, the test item induced up to 2.4- and 2.2 fold, dose related, increases in the number of revertant colonies compared to the solvent control in the first and second experiment respectively.

Strain TA100

In the absence of S9 -mix, the test item induced up to 3.9- and 5.5 -fold, dose related, increases in the number of revertant colonies compared to the solvent control in the first and second experiment respectively. In the presence of S9 -mix, the test item induced up to 1.9- and 2.2 -fold, dose related, increases in the number of revertant colonies compared to the solvent control in the first and second experiment respectively.

Table 1: Mutagenic response of the test item in the Salmonella typhimurium reverse mutation assay. Experiment 1

 Dose (µg/plate)   Mean number of revertant (HIS+) colonies/3 replicate plates (+- S.D.) with two strains of Salmonella typhimurium
   TA98  TA100
      Without S9 -mix
 positive control 589 +/- 29  1089 +/- 99 
 solvent control  17 +/- 5 66 +/- 7 
 3  12 +/- 3 77 +/- 17 
 10  13 +/- 4 68 +/- 4 
 33 12 +/- 2  67 +/- 17 
 100  13 +/- 2  77 +/- 15
 333  15 +/- 3  90 +/- 3
 1000  11 +/- 3 *  121 +/- 12
 3330 MC**   259 +/- 63*
 5000  0 +/- 0*** MC** 

 Dose (µg/plate)   Mean number of revertant (HIS+) colonies/3 replicate plates (+- S.D.) with two strains of Salmonella typhimurium
   TA98  TA100
      With S9 -mix
 positive control 1132 +/- 191  1175 +/- 122 
 solvent control  19 +/- 1 78 +/- 10 
 3  17 +/- 4 101 +/- 2 
 10  19 +/- 3 74 +/- 9 
 33 17 +/- 5  82 +/- 8 
 100  17 +/- 3  87 +/- 6
 333  21 +/- 5  86 +/- 13
 1000  31 +/- 1  93 +/- 11
 3330 37 +/- 6   148 +/- 12
 5000  45 +- 6 75 +- 16

Solvent control; 0.1 ml dimethylsulphoxide

* Bacterial background lawn slightly reduced

** Bacterial background lawn extremely reduced

*** Bacterial background lawn absent

MC: Microcolonies

Table 2: Mutagenic response of the test item in the Salmonella typhimurium reverse mutation assay. Experiment 2

 Dose (µg/plate)   Mean number of revertant (HIS+) colonies/3 replicate plates (+- S.D.) with two strains of Salmonella typhimurium
   TA98  TA100
      Without S9 -mix
 positive control - 1224 +/- 106 
 solvent control - 62 +/- 3 
 500  - 104 +/- 7 
 1000 - 116 +/- 13 
 2000 - 230 +/- 45 
 3000 -  338 +/- 16
 4000 -  34 +/- 24*

 Dose (µg/plate)   Mean number of revertant (HIS+) colonies/3 replicate plates (+- S.D.) with two strains of Salmonella typhimurium
   TA98  TA100
      With S9 -mix
 positive control 1315 +/- 72 1200 +/- 153 
 solvent control 22 +/- 3 69 +/- 6 
 500  33 +/- 1 99 +/- 8 
 1000 37 +/- 3 89 +/- 10 
 2000 47 +/- 8 116 +/- 13 
 3000 43 +/- 5  144 +/- 7
 4000 48 +/- 2  153 +/- 9
 5000  42 +/- 3

* Bacterial background lawn moderately reduced

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that the test substance is mutagenic in the Salmonella typhimurium reverse mutation assay (Strains TA98 and TA100).