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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:
16 July 1993 to 04 Oct 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium 1-amino-4-[[3,5-bis[[(chloroacetyl)amino]methyl]-2,4,6-trimethylphenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate
EC Number:
279-365-5
EC Name:
Sodium 1-amino-4-[[3,5-bis[[(chloroacetyl)amino]methyl]-2,4,6-trimethylphenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate
Cas Number:
80010-51-1
Molecular formula:
C29H28Cl2N4O7S.Na
IUPAC Name:
sodium 1-amino-4-[(3,5-bis{[(chloroacetyl)amino]methyl}-2,4,6-trimethylphenyl)amino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate
Test material form:
solid: particulate/powder
Details on test material:
Test material: Polar blau RLS roh trocken (FAT 21036/C)
Batch No.: 410830.32
Purity: about 90 %
Expiration date: June 1998
Specific details on test material used for the study:
Name: FAT 21036/C
Purity: Approx 90 %

Method

Target gene:
Histidine for Salmonella
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Histidine-auxotrophic strains of Salmonella typhimurium (TA 98, TA 102, TA 1535 and TA 1537) were obtained from Prof. B.
Ames, Berkeley, CA., U.S.A
Strain TA 100 was obtained from Dr. M.Schiipbach, Hofmann-La Roche Ltd., Basle, Switzerland.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat-liver post mitochondrial supernatant (S9 fraction): Rat-liver microsomal fraction S9 was prepared in advance from male RAI rats (Tif: RAIf[SPF]), reared at the Animal Farm of CIBA GEIGY, Sisseln, Switzerland. The animals (150-250 g) were treated with Aroclor 1254 (500 mg/kg, i.p.) 5 days prior to sacrifice. The livers were homogenized with 3 volumes of 150 mM KCl and the 9000x g supernatant (S9) was stored at approximately -80 °C for no longer than one year. The protein contents of the S9 fractions were 33.6 and 30.2 mg/ml.
Test concentrations with justification for top dose:
Range in the cytotoxicity test; 20.6, 61.7, 185.1, 555.5, 1666.6, 5000 µg/plate
Range in the mutagenicity test: 61.7, 185.1, 555.5, 1666.6, 5000 µg/plate
Vehicle / solvent:
Bidistilled water
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
For TA 100 & TA 1535 without microsomal activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
For TA 102 without microsomal activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
For TA 98 without microsomal activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
For TA 1537 without microsomal activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2- aminoanthracene
Remarks:
For TA 100, TA 102, TA 98& TA 1537 with microsomal activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
For TA 1535 with microsomal activation
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments : Two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium: in agar (plate incorporation)

METHODS FOR MEASUREMENT OF CYTOTOXICITY : Background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY : Colonies were counted electronically with an Artek counter
Evaluation criteria:
Assay acceptance criteria:
A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response. In either case the final decision is based on the scientific judgement of the study director.

Criteria for a positive response:
The test substance is considered to be mutagenic in this test system if the following conditions are met:
At least a reproducible meaningful increase of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the following strains: S. typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537. Generally a concentration-related effect should be demonstrable.
Statistics:
In deviation to the OECD guideline. a statistical analysis was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended. No appropriate statistical method is available.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

RANGE-FINDING/SCREENING STUDIES


Six concentrations of Polar blau RLS roh trocken (FAT 21036/C) ranging from 20.6 to 5000 µg/plate were tested with strain S. typhimurium TA 100 to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without microsomal activation. The numbers of revertant colonies was not reduced. Normal back-ground growth was observed at all concentrations. The test material exerted no toxic effect on the growth of the bacteria. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 µg/plate without and with activation.


 


Mutagenicity test, original experiment


In the original experiment carried out without metabolic activation, treatment of strain TA 102 with Polar blau RLS roh trocken (FAT 2103 6/C) led to a marginal increase in the number of backmutants at the concentrations of 1666.7 and 5000 µg/plate. No effects were observed with the other strains. In the experiment with activation performed on strain TA 102, a marginal increase in the number of revertants occurred at the concentration of 555.6 µg/plate only. No effects were observed with the other strains.


 


Mutagenicity test, confirmatory experiment


In the confirmatory experiment performed without microsomal activation, after treatment with Polar blau RLS roh trocken (FAT 21036/C), no increase in the incidence of histidine-prototrophic mutants was observed in comparison with the negative control. The effect observed in the original experiment on strain TA 102 could not be reproduced. In the experiment with activation performed on strain TA 102, a marginal increase in the number of back-mutants occurred at the concentration of 1666.7 µg/plate only. Again, no effects were observed with the other strains. In the mutagenicity tests without and with metabolic activation, normal back-ground growth was observed. The numbers of revertant colonies was not reduced. The test material exerted no toxic effect on the growth of the bacteria. The various mutagens, promutagens, sterility checks, sensitivity and resistance tests, etc., employed to ensure the test system was acceptable, all produced results within our established limits. There were no known circumstances or occurrences in this study that were considered to have affected the quality or integrity of the data.


 


SUMMARY OF THE MUTAGENICITY EXPERIMENTS


Original experiments without metabolic activation


 








































Strain



Treatment



Mean counts



TA 100



Negative control



126.3



61.7284 µg/plate



141.3



185.1852 µg/plate



155.0



555.5556 µg/plate



143.0



1666.6667 µg/plate



144.3



5000.0000 µg/plate



108.7



Positive control



1320.3



 








































Strain



Treatment



Mean counts



TA 1535



Negative control



13.7



61.7284 µg/plate



14.7



185.1852 µg/plate



11.7



555.5556 µg/plate



11.7



1666.6667 µg/plate



14.7



5000.0000 µg/plate



11.33



Positive control



1100.7



 


 








































Strain



Treatment



Mean counts



TA 98



Negative control



15.0



61.7284 µg/plate



16.0



185.1852 µg/plate



15.3



555.5556 µg/plate



21.0



1666.6667 µg/plate



14.7



5000.0000 µg/plate



13.0



Positive control



1582.7



 








































Strain



Treatment



Mean counts



TA 1537



Negative control



9.7



61.7284 µg/plate



8.0



185.1852 µg/plate



9.0



555.5556 µg/plate



4.3



1666.6667 µg/plate



5.0



5000.0000 µg/plate



5.0



Positive control



2091.7



 








































Strain



Treatment



Mean counts



TA 102



Negative control



237.0



61.7284 µg/plate



240.0



185.1852 µg/plate



273.3



555.5556 µg/plate



327.3



1666.6667 µg/plate



394.3



5000.0000 µg/plate



378.0



Positive control



1130.3



 


 


SUMMARY OF THE MUTAGENICITY EXPERIMENTS


Original experiments with metabolic activation


 








































Strain



Treatment



Mean counts



TA 100



Negative control



135.3



61.7284 µg/plate



130.3



185.1852 µg/plate



142.7



555.5556 µg/plate



173.0



1666.6667 µg/plate



159.3



5000.0000 µg/plate



144.3



Positive control



1389.7



 








































Strain



Treatment



Mean counts



TA 1535



Negative control



17.0



61.7284 µg/plate



17.7



185.1852 µg/plate



14.7



555.5556 µg/plate



15.3



1666.6667 µg/plate



14.3



5000.0000 µg/plate



10.7



Positive control



339.7



 


 








































Strain



Treatment



Mean counts



TA 98



Negative control



34.0



61.7284 µg/plate



33.7



185.1852 µg/plate



29.3



555.5556 µg/plate



31.3



1666.6667 µg/plate



23.3



5000.0000 µg/plate



23.7



Positive control



2207.3



 








































Strain



Treatment



Mean counts



TA 1537



Negative control



7.3



61.7284 µg/plate



9.0



185.1852 µg/plate



12.0



555.5556 µg/plate



11.3



1666.6667 µg/plate



7.3



5000.0000 µg/plate



4.0



Positive control



205.7



 








































Strain



Treatment



Mean counts



TA 102



Negative control



244.7



61.7284 µg/plate



348.3



185.1852 µg/plate



295.3



555.5556 µg/plate



389.3



1666.6667 µg/plate



349.0



5000.0000 µg/plate



354.0



Positive control



1431.7



 


 


SUMMARY OF THE MUTAGENICITY EXPERIMENTS


Confirmatory experiments without metabolic activation


 








































Strain



Treatment



Mean counts



TA 100



Negative control



173.3



61.7284 µg/plate



195.7



185.1852 µg/plate



200.3



555.5556 µg/plate



174.3



1666.6667 µg/plate



194.3



5000.0000 µg/plate



183.3



Positive control



1345.0



 








































Strain



Treatment



Mean counts



TA 1535



Negative control



19.0



61.7284 µg/plate



14.7



185.1852 µg/plate



20.7



555.5556 µg/plate



18.0



1666.6667 µg/plate



15.7



5000.0000 µg/plate



15.0



Positive control



1187.7



 


 








































Strain



Treatment



Mean counts



TA 98



Negative control



28.3



61.7284 µg/plate



28.7



185.1852 µg/plate



24.7



555.5556 µg/plate



24.7



1666.6667 µg/plate



27.7



5000.0000 µg/plate



23.0



Positive control



1935.0



 








































Strain



Treatment



Mean counts



TA 1537



Negative control



10.3



61.7284 µg/plate



12.0



185.1852 µg/plate



14.7



555.5556 µg/plate



10.0



1666.6667 µg/plate



9.7



5000.0000 µg/plate



6.7



Positive control



2315.0



 








































Strain



Treatment



Mean counts



TA 102



Negative control



336.7



61.7284 µg/plate



340.3



185.1852 µg/plate



391.7



555.5556 µg/plate



374.3



1666.6667 µg/plate



419.3



5000.0000 µg/plate



357.7



Positive control



1961.0



 


 


SUMMARY OF THE MUTAGENICITY EXPERIMENTS


Confirmatory experiments with metabolic activation


 








































Strain



Treatment



Mean counts



TA 100



Negative control



184.7



61.7284 µg/plate



208.3



185.1852 µg/plate



226.7



555.5556 µg/plate



219.3



1666.6667 µg/plate



226.0



5000.0000 µg/plate



213.7



Positive control



1881.0



 








































Strain



Treatment



Mean counts



TA 1535



Negative control



17.3



61.7284 µg/plate



24.3



185.1852 µg/plate



20.7



555.5556 µg/plate



16.3



1666.6667 µg/plate



25.3



5000.0000 µg/plate



19.7



Positive control



378.7



 


 








































Strain



Treatment



Mean counts



TA 98



Negative control



42.7



61.7284 µg/plate



43.0



185.1852 µg/plate



49.3



555.5556 µg/plate



47.0



1666.6667 µg/plate



33.7



5000.0000 µg/plate



33.7



Positive control



2659.0



 








































Strain



Treatment



Mean counts



TA 1537



Negative control



14.7



61.7284 µg/plate



9.3



185.1852 µg/plate



12.3



555.5556 µg/plate



15.3



1666.6667 µg/plate



9.0



5000.0000 µg/plate



6.7



Positive control



414.7



 








































Strain



Treatment



Mean counts



TA 102



Negative control



331.7



61.7284 µg/plate



369.3



185.1852 µg/plate



379.7



555.5556 µg/plate



427.3



1666.6667 µg/plate



496.3



5000.0000 µg/plate



458.7



Positive control



1723.3



 

Applicant's summary and conclusion

Conclusions:
FAT 21036/C was not mutagenic in this bacterial reverse mutation assay.
Executive summary:

A study was performed to investigate the potential of FAT 21036/C to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. This test was conducted in accordance with OECD test guideline 471. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations:


Range in the cytotoxicity test; 20.6, 61.7, 185.1, 555.5, 1666.6, 5000 µg/plate


The concentration range of FAT 21036/C to be tested in the mutagenicity test was determined in a preliminary toxicity test. Thus, the test material was tested for mutagenic effects without and with metabolic activation at five concentrations in the range 61.7, 185.1, 555.5, 1666.6, 5000 µg/plate.


 


Mutagenicity test, original experiment


In the original experiment carried out without metabolic activation, treatment of strain TA 102 with Polar blau RLS roh trocken (FAT 2103 6/C) led to a marginal increase in the number of backmutants at the concentrations of 1666.7 and 5000 µg/plate. No effects were observed with the other strains. In the experiment with activation performed on strain TA 102, a marginal increase in the number of revertants occurred at the concentration of 555.6 µg/plate only. No effects were observed with the other strains.


 


Mutagenicity test, confirmatory experiment


In the confirmatory experiment performed without microsomal activation, after treatment with Polar blau RLS roh trocken (FAT 21036/C), no increase in the incidence of histidine-prototrophic mutants was observed in comparison with the negative control. The effect observed in the original experiment on strain TA 102 could not be reproduced. In the experiment with activation performed on strain TA 102, a marginal increase in the number of back-mutants occurred at the concentration of 1666.7 µg/plate only. Again, no effects were observed with the other strains. In the experiment without and with metabolic activation no toxic effect of the test material on the growth of the bacteria was observed. A marginal increase in backmutants was reported with strain TA 102 with metabolic activation at 555.6 µg/plate only in the original experiment and at 1666.7 µg/plate only in the confirmatory experiment. This marginal effect seen was regarded as not significant as the increase was seen at two different concentrations during original and confirmatory experiments, without being seen at higher concentrations in these experiments. Thus, the effect seen can not be considered as dose-dependent or concentration driven. Hence, based on the results of these experiments and on standard evaluation criteria, it is concluded that FAT 21036/C was not mutagenic in this bacterial reverse mutation assay.