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Toxicological information

Genetic toxicity: in vitro

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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in accordance with GLP; low purity of the test substance (72.1 %)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 3-(4-Methoxyphenyl)-3-methoxypropionsaueremethylester, technical grade
- Substance No.: 93/98
- Batch No.: Ansatz 165
- Purity: 71.2 %.
- Date of manufacturing: 22 Apr 1993
- Physical state: solid, brown; after heating at about30 °C: liquid brown
- Storage condition of test material: REFRIGERATOR, EXCLUSION OF LIGHT AND AIR (UNDER NITROGEN), EXCLUSION OF MOISTURE

Method

Target gene:
Histidine operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9-mix
Test concentrations with justification for top dose:
- 1st experiment: 20, 100, 500, 2500, 5000 µg/plate
- 2nd, 3rd, 4th and 5th experiment: 1000, 2000, 3000, 4000, 5000 µg/plate
Vehicle / solvent:
Vehicle used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: With S9-mix: 2.5 µg/plate 2-aminoanthracene for the strains TA 100, TA 98, TA 1537 and TA 1535; without S9-mix: 5 µg/plate N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for the strains TA 100 and TA 1535, 10 µg/plate 4-nitro-o-phenylenediamine (NPD) for
Remarks:
all positive control substances dissolved in DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- standard plate test

DURATION
- Preincubation period: no
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

USED STRAINS
1st experiment: strains TA 1535, TA 100, TA 1537, TA 98
2nd, 3rd, 4th and 5th experiments: strain TA1537

DETERMINATION OF CYTOTOXICITY
- decrease in number of his-revertants
- reduction of titer determination; titer only determined in the experiments with S-9 mix both without test substance (vehicle only) and after adding the two highest doses of test substance




Evaluation criteria:
In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results

Results and discussion

Test resultsopen allclose all
Species / strain:
other: S. typhimurium TA, 1535, TA 98, TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 2500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 2500 µg/plate depending on the experiment
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: S. typhimurium TA, 1535, TA 98, TA 100
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Cytotoxicity: A weakly bacteriotoxic effect (slight decrease in the number of his + revertants and a reduced titer) was observed

depending on the strain and test conditions at doses >= 2500 µg/plate.

Mutagenicity:

Tests without S-9 mix:

The strains TA 1535, TA 100 and TA 98 showed no increase in the number of his+ revertants.

The strain TA 1537 showed occasionally some increased colony numbers in the 1st (factor 0.6 -1.5), 3rd (factor 0.5 -1.6),

4th (0.7 - 1.5) and 5th (factor 0.7 -1.4) experiment. The evaluation criteria indicated a positive reaction, when among other things a

doubling of the spontaneous mutation rate was observed. So the findings were stated as not relevant. A positive reaction was

observed in the 2nd experiment from 1000 - 5000 µg/plate (factor 1.8 - 4.4).

Tests with S-9 mix:

The strains TA 1535, TA 100 and TA 98 showed no increase in the number of his+ revertants.

The strain TA 1537 showed no increase in the number of his+ revertants in the 3rd and 4th experiments. Some not relevant, slightly

increased colony numbers were observed in the 1st experiment (factor 1.0 -1.9). A positive reaction was observed in the 2nd

experiment at 2000 - 4000 µg (factor 2.1 - 3.4) and in the 5th experiment at 3000 - 5000 µg/plate (factor 1.9 -3.1).

The positive controls showed the expected increase in his+ revertants/plate.

Results:

Dose µg/plate metabolic activation  TA98   TA100   TA1535   TA1537 
Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3
0 + 44 34 47 144 107 114 13 11 17 10 10 13
20 + 51 37 53 112 121 128 13 20 15 10 11 11
100 + 43 46 43 125 138 136 15 16 15 17 7 10
500 + 37 41 37 132 127 127 20 14 14 19 10 20
2500 + 39 37 35 134 125 120 13 15 17 19 23 20
5000 + 25 19 25 117 118 132 20 23 15 19 16 16
           
2.5 µg 2-Aminoanthracene + 670 586 676 970 970 930 111 88 96 82 92 114
           
0 - 38 29 39 114 115 89 16 19 23 13 12 14
20 - 21 21 26 107 113 122 20 20 20 6 8 8
100 - 43 28 30 100 148 121 27 20 21 6 10 9
500 - 29 32 23 118 113 116 23 26 21 20 10 9
2500 - 27 39 30 108 128 121 25 22 30 20 14 23
5000 - 42 36 31 117 71 80 29 33 26 18 19 19
           
5 µg MNNG -     873 820 770 920 865 660
10 µg 4-Nitro-o-phenylendiamin - 1054 1074 982    
100 µg 9-Aminoacridiniumchloride monohydrate -                   602 804 843

Dose µg/plate metabolic activation  TA1537   TA1537   TA1537   TA1537 
Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3
0 + 12 16 18 21 8 12 20 15 10 9 10 12
20 + 13 10 22 3 15 10 11 15 14 7 12 15
100 + 30 33 33 22 12 16 15 7 12 15 6 15
500 + 46 43 52 21 11 10 7 12 10 23 12 25
2500 + 47 51 60 13 12 10 12 10 15 29 20 15
5000 + 19B 25B 30B 2B 1B B 24 12 18 29 28 38
             
2.5 µg 2-Aminoanthracene + 87 87 104 78 89 92 105 110 100 95 101 112
             
0 - 10 10 12 12 6 14 11 12 15 10 8 12
20 - 22 17 20 18 6 9 14 14 10 11 8 16
100 - 21 36 48 17 15 15 14 4 13 8 10 11
500 - 49 55 37 15 16 20 9 19 10 10 8 5
2500 - 34 39 48 10 14 11 16 4 7 4 6 10
5000 - 33 42 33 6B 4B B 26 18 14 8 16 18
             
100 µg 9-Aminoacridiniumchloride monohydrate - 418 390 523 490 533 716 659 512 709 342 264 253

B: reduced backround

Applicant's summary and conclusion