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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:
17 Jan - 4 Feb 1990
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:
1990
Report Date:
1990

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his 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:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Dose range finding test: 5, 50, 500 and 5000 µg/plate with and without metabolic activation
Main test: 50, 150, 500, 1500, 5000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 9-Aminoacridine (80 µg/pltae ) for TA1537; N-Ethyl-N'-nitrosoguanidine (3 µg/plate) for TA100, (5 µg/plate) for TA1535; 2-Nitrofluorene (1 µg/plate) for TA98.
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: With S9: 2-Aminoanthracene (0.5 µg/plate) for TA98, (1 µg/plate) for TA100, (2 µg/plate) for TA1535 and TA1537
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

- Preincubation period: 1 h, 37 °C
- Exposure duration: 24 h (range finding test), 72 h (main test), 37 °C

NUMBER OF REPLICATIONS: triplicates, each in two experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER:
- S9: added at 3% (main test) and 10% (range finding test and main test). In a rerun S9 of the main test S9 was added at 10% and 30%, respectively
Evaluation criteria:
The test substance is considered to show evidence of mutagenic activity, if treatment with a test material produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix.
The test substance is considered to show no evidence of mutagenic activity, if treatment with a test material does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at 5000µg/plate for all strains in the presence and absence of S9
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Results main experiment:

Experiment 1:

Dose Level (µg/plate) Liver S-9 mix TA 1535 TA1537 TA98 TA100
mean  SD mean  SD mean  SD mean  SD
5000 - 10 1.0 10 2.1 19 0.0 64 5.6
1500 - 11 1.7 9 0.6 23 3.0 90 1.0
500 - 14 2.5 10 2.1 20 1.5 96 2.5
150 - 12 2.0 9 1.5 22 3.1 102 2.6
50 - 11 1.0 9 0.6 21 2.0 101 3.1
0 - 17 1.0 10 0.6 24 5.0 104 4.5
Solvent - 15 1.0 10 1.0 26 4.0 112 2.5
ENNG (5 µg/plate) - 458 67.1            
9-AC (80 µg/plate) -     1108 51.9        
NF (1 µg/plate) -         85 9.0    
ENNG (3 µg/plate) -             349 44.3
                   
5000 3% 12 1.5 10 0.6 24 2.1 72 1.5
1500 3% 11 4.4 10 1.2 21 2.1 92 2.0
500 3% 14 1.0 9 1.0 19 1.0 99 6.1
150 3% 11 1.0 8 1.0 22 2.6 101 5.0
50 3% 13 1.0 10 1.2 20 0.6 100 2.0
0 3% 18 1.7 10 2.5 17 2.1 99 6.4
Solvent 3% 11 2.5 9 1.0 20 1.2 102 4.5
AA (0.5 µg/plate) 3%         161 20.0    
AA (1 µg/plate) 3%             295 21.6
AA (2 µg/plate) 3% 56 3.8 80 11.5        
                   
5000 10% 12 1.5 10 2.1 22 1.5 73 1.0
1500 10% 11 1.2 9 1.0 20 1.0 93 4.2
500 10% 14 2.5 12 2.1 20 3.8 97 7.6
150 10% 15 2.8 10 1.5 22 3.2 101 2.3
50 10% 11 2.5 10 1.5 21 1.4 108 2.0
0 10% 14 1.0 8 1.5 23 3.6 104 10.2
Solvent 10% 14 1.5 11 1.5 24 3.2 101 2.3
AA (0.5 µg/plate) 10%         143 11.0    
AA (1 µg/plate) 10%             353 10.1
AA (2 µg/plate) 10% 57 7.6 70 8.0        

Experiment 2:

Dose Level (µg/plate) Liver S-9 mix TA 1535 TA1537 TA98 TA100
mean  SD mean  SD mean  SD mean  SD
5000 - 10 3.2 10 1.0 19 3.1 82 6.1
1500 - 10 1.0 14 0.6 21 0.0 104 7.5
500 - 9 0.6 10 2.5 21 2.5 100 11.2
150 - 12 1.0 11 2.6 19 1.0 95 5.6
50 - 10 1.0 14 1.5 22 0.6 100 2.5
0 - 9 0.6 11 1.2 23 3.0 89 7.6
Solvent - 11 0.6 12 3.2 22 1.2 100 14.5
ENNG (5 µg/plate) - 439 48.8            
9-AC (80 µg/plate) -     x x        
NF (1 µg/plate) -         80 8.0    
ENNG (3 µg/plate) -             424 56.0
                   
5000 10% 11 2.0 17 3.0 19 2.1 92 4.0
1500 10% 14 2.0 16 0.6 22 2.5 100 12.7
500 10% 11 2.1 13 1.2 20 0.0 107 3.1
150 10% 10 0.6 15 1.5 22 1.0 108 4.0
50 10% 12 2.5 12 2.1 23 1.5 107 3.1
0 10% 10 2.0 16 4.2 20 2.0 100 10.4
Solvent 10% 10 1.7 15 2.5 22 2.5 105 10.5
AA (0.5 µg/plate) 10%         87 1.2    
AA (1 µg/plate) 10%             358 48.0
AA (2 µg/plate) 10% 71 18.8 68 12.8        
                   
5000 30% 9 0.6 17 1.2 19 3.5 101 7.6
1500 30% 9 0.6 16 2.1 23 2.0 106 5.3
500 30% 10 1.5 14 2.0 21 3.1 107 4.5
150 30% 11 0.6 14 2.3 24 0.0 110 3.0
50 30% 12 1.7 14 2.9 27 1.0 110 9.0
0 30% 12 1.0 16 0.6 21 2.1 118 8.6
Solvent 30% 12 2.1 17 2.5 21 1.2 110 10.7
AA (0.5 µg/plate) 30%         83 6.1    
AA (1 µg/plate) 30%             299 30.2
AA (2 µg/plate) 30% 74 9.3 61 8.5        

SD: standard deviation

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

9 -AC: 9 -aminoacridine

AA: 2 -aminoanthracene

NF: 2 -nitrofluorene

x: too many colonies to count accurately

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative