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Diss Factsheets

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:
20 July, 1993 to 22 Sptember, 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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
None
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Disodium[4-{[5-chloro-2-(hydroxy-O)-3-nitrophenyl]diazenyl-N1}-3-methyl-1-phenyl-1H-pyrazol-5-olato(2-)-O][3-{[1-(3-chlorophenyl)-5-(hydroxy-O)-3-methyl-1H-pyrazol-4-yl]diazenyl-N1}-4-(hydroxy-kO)-5-nitrobenzenesulfonato(3-)]chromate(2-)
Cas Number:
72017-66-4
Molecular formula:
C32H19Na2Cl2CrN10O11S
IUPAC Name:
Disodium[4-{[5-chloro-2-(hydroxy-O)-3-nitrophenyl]diazenyl-N1}-3-methyl-1-phenyl-1H-pyrazol-5-olato(2-)-O][3-{[1-(3-chlorophenyl)-5-(hydroxy-O)-3-methyl-1H-pyrazol-4-yl]diazenyl-N1}-4-(hydroxy-kO)-5-nitrobenzenesulfonato(3-)]chromate(2-)
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Name: FAT 20'202/E
Batch No.: Op. 161
Aggregate State at RT: solid
Colour:light red
Purity: 70.1 (active ingredient)
Analysis: cf. information in sponsor's file
Stability: Pure: until March, 1998 In solvent: if necessary will be performed by the sponsor at a later date.
Storage: room temperature
Expiration Date: March, 1998

Method

Target gene:
Histidine auxotrophs
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: The Salmonella typhimurium histidine (his) reversion system measures his- —> his+ reversions.
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from rat liver
Test concentrations with justification for top dose:
33.3; 100; 333.3; 1000; 2500; and 5000 µg/plate
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:
Without metabolic activation - TA 1535 and TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
Without metabolic activation - TA 1537 and TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activaton - TA 102
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With metabolic activation - TA 1535, TA 1537, TA 98, TA 100, TA 102
Details on test system and experimental conditions:
Without metabolic activation
TA 1535, TA 100, TA 102 - double distilled water
TA 15377, TA 98 - DMSO


With metabolic activation
TA 1535, TA 1537, TA 98, TA 100, TA 102 - DMSO

THE TEST SYSTEM
Characterisation of the Salmonella typhimurium Strains.The strains are derived from S. typhimurium strain LT2 and due to a mutation in the histidine locus are histidine dependent. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98, TA 100 and TA 102 the R-factor plasmid pKM 101 carries the ampicillin resistance marker. The strain TA 102 does not contain the uvrB"-mutation. Additionally TA 102 contains the multicopy plasmid pAQl, which carries the hisG428 mutation and a tetracycline resistance gene. TA 102 contains the ochre mutation in hisG gene.

Storage
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.

Precultures
From the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. This nutrient medium contains per litre:
8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt)
5 g NaCl (MERCK, D-64293 Darmstadt)
The bacterial culture was incubated in a shaking water bath for 10 hours at 37 °C.

Selective Agar
2.0 % Vogel-Bonner-Glucose-Minimal-Agar was used as selective agar. Each petri dish was filled with 20 ml of this nutrient medium. Sterilisations were performed at 121 °C in an autoclave.

Overlay Agar
The overlay agar contains per litre:
6.0 g Merck Agar Agar*
6.0 g NaCl*
10.5 mg L-histidine x HCl x H20*
12.2 mg biotin*

Sterilisations were performed at 121 °C in an autoclave.

MAMMALIAN MICROSOMAL FRACTION S9 MIX
S9 (Preparation by C CR) The S9 liver microsomal fraction was obtained from the liver of 8-12 weeks old male Wistar rats, strain WU (Charles River Wiga GmbH, D-97633 Sulzfeld, F.R.G.; weight approx. 150 - 200 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 (Antechnika, D-76275 Ettlingen, F.R.G.) in olive oil 5 days previously. After cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate,
diluted 1+3 in KCl was centrifuged cold at 9,000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2, 3 or 5 ml and stored at -70 °C. Small numbers of the ampoules are kept at -20 °C for only several weeks before use. The standardisation of the protein content was made using the analysis kit of Bio-Rad Laboratories, D-80939 München:Bio-Rad protein assay, Catalogue 500 000 6 (6).The protein concentration in the S9 preparation was 43.8 mg/ml
(lot 010293) in the pre-experiment as well as in experiment I, and 45.0 mg/ml (lot 190793) in experiment II.

S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cof actor solution. The amount of S9 supernatant was 15 % v/v. The composition of the cofactor solution was concentrated to yield the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The
S9 mix preparation was performed according to Ames et al.(2).


PRE-EXPERIMENT FOR TOXICITY
To evaluate the toxicity of the test article a prestudy was performed with strains TA 9 8 and TA 100. 8 concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described below for the experiment I (plate incorporation test). Toxicity of the test article may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.


* (MERCK, D-64293 Darmstadt)
Sterilisations were performed at 121 °C in an autoclave.
Rationale for test conditions:
None
Evaluation criteria:
The generally accepted conditions for the evaluation of the results are:
corresponding background growth on both negative control and test plates normal range of spontaneous reversion rates.

Due to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time.

A test article is considered as positive if either a dose related and reproducible increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.

A test article producing neither a dose related and reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

A significant response is described as follows:
A test article is considered as mutagenic if in strain TA 100 and TA 102 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.

Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.
Statistics:
No appropriate statistical method is available.

Results and discussion

Test results
Key result
Species / strain:
other: 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:
not specified
Positive controls validity:
valid
Additional information on results:
None
Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

Test article: FAT 20202/E Lanacron Rot S-3G roh feucht


S9 mix from: rat liver (Batch R 010293)


Test strain: TA 1535


 
















































































































































































































Doseµg/plate (active ingredient)PLATE  Revertants/plate  
123meanS.D.Factor+
without S9 mix
Negative Control81413123.2 
Solvent Control221910176.21
 33.31213141310.8
 10081016114.20.7
 333.3171213142.60.8
 100010719126.20.7
 2500312210.1
 5000000000
Positive Control Sodium azide (10µg/plate)10661051106910629.662.5
with S9 mix
Negative Control201020175.8 
Solvent Control241521204.61
 33.3712792.90.4
 100141721173.50.9
 333.3262435282.91.4
 1000141518162.10.8
 2500121917163.60.8
 500011127102.60.5
Positive Control 2-Aminoanthracene (2.5µg/plate)139151343211114.510.6
 
 

 


 


Test article: FAT 20202/E Lanacron Rot S-3G roh feucht


S9 mix from: rat liver       


Test strain: TA 1537


 


 
















































































































































































































Doseµg/plate (active ingredient)PLATE  Revertants/plate  
123meanS.D.Factor+
without S9 mix
Negative Control91613133.5 
Solvent Control1210693.11
 33.35101082.90.9
 1001311151321.4
 333.318811125.11.3
 100081212112.31.1
 250091111101.21.1
 500043330.60.4
Positive Control 4-nitor-o-phenylene diamine (10µg/plate)929185893.89.6
with S9 mix
Negative Control181419172.6 
Solvent Control261720214.61
 33.3232121221.21
 100191727244.61.2
 333.3232032256.21.2
 1000292321244.21.2
 2500241722213.61
 5000251527226.41.1
Positive Control 2-Aminoanthracene (2.5µg/plate)9591939324.4
 
 

 


 


Test article: FAT 20202/E Lanacron Rot S-3G roh feucht


S9 mix from : rat liver (Batch R 010293)


Test strain : TA 98


 


 
















































































































































































































Doseµg/plate (active ingredient)PLATE  Revertants/plate  
123meanS.D.Factor+
without S9 mix
Negative Control262736305.5 
Solvent Control231928234.51
 33.3213238308.61.3
 1001327212070.9
 333.3201920200.60.8
 1000151217152.50.6
 250072752.90.2
 500010310840.3
Positive Control      4-nitorphenylene diamine (10µg/plate)81780773678744.233.7
with S9 mix
Negative Control415336438.7 
Solvent Control394155458.71
 33.3465439467.51
 100404449444.51
 333.3393332353.80.8
 1000242926262.50.6
 2500203123255.70.5
 5000101617143.80.3
Positive Control 2-Aminoanthracene (2.5µg/plate)5835024985284811.7
 
 

 


 


Test article: FAT 20202/E Lanacron Rot S-3G roh feucht


S9 mix from : rat liver (Batch R 010293)


Test strain : TA 100


 


 
















































































































































































































Doseµg/plate (active ingredient)PLATE  Revertants/plate  
123meanS.D.Factor+
without S9 mix
Negative Control1011179610511 
Solvent Control1159712011112.11
 33.3711078889180.8
 100110101991035.90.9
 333.38578998710.70.8
 1000828179811.50.7
 2500971168610015.20.9
 500099981009910.9
Positive Control      sodium azide (10µg/plate)75573766671947.16.5
with S9 mix
Negative Control18314615416119.5 
Solvent Control1591631661633.51
 33.31851751781795.11.1
 1001741671591677.51
 333.31641791671707.91
 10001801941971909.11.2
 25001952031861958.51.2
 500022819919220619.11.3
Positive Control     2-Aminoanthracene (2.5µg/plate)10078841090994103.66.1
 
 

 


 


Test article: FAT 20202/E Lanacron Rot S-3G roh feucht


S9 mix from : rat liver (Batch R 010293)


Test strain : TA 102


 


 
















































































































































































































Doseµg/plate (active ingredient)PLATE  Revertants/plate  
123meanS.D.Factor+
without S9 mix
Negative Control15614316315410.7 
Solvent Control16113813614513.91
 33.315315317616113.31.1
 10017217812215730.71.1
 333.32021951941974.41.4
 10001781741791772.61.2
 25001078710910112.20.7
 50001414141400.1
Positive Control   methyl methane sulfonate                (1.0µl/plate)106610401154108759.77.5
with S9 mix
Negative Control23926721524026 
Solvent Control2662802682717.61
 33.328831329930012.51.1
 10021323120521613.30.8
 333.329528832130117.41.1
 100025029423726029.91
 250022628532928051.71
 500027737634633350.81.2
Positive Control      2-Aminoanthracene (10µg/plate)89386883486529.63.2
 
 

 


 


PRE-INCUBATION TEST


 


Test article: FAT 20202/E Lanacron Rot S-3G roh feucht


S9 mix from : rat liver (Batch R 190793)


Test strain : TA 1535


 


 
















































































































































































































Doseµg/plate (active ingredient)PLATE  Revertants/plate  
123meanS.D.Factor+
without S9 mix
Negative Control14613114.4 
Solvent Control51213104.41
 33.313116103.61
 10034752.10.5
 333.3711583.10.8
 1000109111011
 250067461.50.6
 5000000000
Positive Control Sodium azide (10µg/plate)5264940340294.934
with S9 mix
Negative Control222124221.5 
Solvent Control223338318.21
 33.3132529228.30.7
 100211829235.70.7
 333.3122517186.60.6
 10002621332760.9
 25002318262240.7
 5000141417151.70.5
Positive Control 2-Aminoanthracene (2.5µg/plate)4124264144177.613.5
 
 

 


 


Test article: FAT 20202/E Lanacron Rot S-3G roh feucht


S9 mix from : rat liver (Batch R 190793)


Test strain : TA 1537


 


 
















































































































































































































Doseµg/plate (active ingredient)PLATE  Revertants/plate  
123meanS.D.Factor+
without S9 mix
Negative Control17119124.2 
Solvent Control141016133.11
 33.3105772.50.6
 1009914112.90.8
 333.3814111130.8
 1000131121155.31.1
 250033430.60.3
 5000000000
Positive Control 4-nitor-o-phenylene diamine (10µg/plate)1091031111084.28.1
with S9 mix
Negative Control11108101.5 
Solvent Control141113131.51
 33.361319136.51
 100171110133.81
 333.315911123.10.9
 1000121114121.51
 2500131215131.51.1
 500091316133.51
Positive Control 2-Aminoanthracene (2.5µg/plate)747587797.26.2
 
 

 


 


Test article: FAT 20202/E Lanacron Rot S-3G roh feucht


S9 mix from : rat liver (Batch R 190793)


Test strain : TA 98


 


 
















































































































































































































Doseµg/plate (active ingredient)PLATE  Revertants/plate  
123meanS.D.Factor+
without S9 mix
Negative Control262821253.6 
Solvent Control1724171941
 33.3242516224.91.1
 1001319251961
 333.3223034296.11.5
 1000111822175.60.9
 250010109100.60.5
 5000000000
Positive Control      4-nitorphenylene diamine (10µg/plate)55751651653023.727.4
with S9 mix
Negative Control384636405.3 
Solvent Control454430408.41
 33.3445847507.41.3
 1005459475361.3
 333.34750535031.3
 10003763585313.81.3
 25002925332940.7
 50002227152160.5
Positive Control 2-Aminoanthracene (2.5µg/plate)42048640943841.611.1
 
 

 


 


Test article: FAT 20202/E Lanacron Rot S-3G roh feucht


S9 mix from : rat liver (Batch R 190793)


Test strain : TA 100


 


 
















































































































































































































Doseµg/plate (active ingredient)PLATE  Revertants/plate  
123meanS.D.Factor+
without S9 mix
Negative Control859477858.5 
Solvent Control1038911210111.61
 33.375103838714.40.9
 100647476716.40.7
 333.3694880765.90.7
 100010181758613.60.8
 25006073977718.80.8
 50001659815233.10.5
Positive Control      sodium azide (10µg/plate)407430474437344.3
with S9 mix
Negative Control1731721861777.8 
Solvent Control1581531601573.61
 33.317815213615521.21
 1001681511511579.81
 333.31841861691809.31.1
 100016517717017161.1
 250017917112115731.41
 50001261311311292.90.8
Positive Control     2-Aminoanthracene (2.5µg/plate)86993793591438.75.8
 
 

 


Test article: FAT 20202/E Lanacron Rot S-3G roh feucht


S9 mix from : rat liver (Batch R 190793)


Test strain : TA 102


 


 
















































































































































































































Doseµg/plate (active ingredient)PLATE  Revertants/plate  
123meanS.D.Factor+
without S9 mix
Negative Control1591571561571.5 
Solvent Control15218217316915.41
 33.314215417115614.60.9
 1001351351301332.90.8
 333.317917320618617.61.1
 10001791781861814.41.1
 25002653414013.50.2
 5000000000
Positive Control   methyl methane sulfonate                (1.0µl/plate)967924881924435.5
with S9 mix
Negative Control25127126926411 
Solvent Control23620023622420.81
 33.32812652792758.71.2
 100238217261239221.1
 333.321320225722429.11
 100021027227125135.51.1
 250025820520322231.21
 500025423022023517.51
Positive Control      2-Aminoanthracene (10µg/plate)63163969365433.72.9
 
 

Applicant's summary and conclusion

Conclusions:
FAT 20202/E was determined to be non-mutagenic in the bacterial reverse mutation assay.
Executive summary:

FAT 20202/E was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium which included TA 1535, TA 1537, TA 98, TA 100, and TA 102. The investigations were performed with the following concentrations of the trial substance with and without microsomal activation: 33.3; 100; 333.3; 1000; 2500; and 5000 µg/plate


The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. In both experiments toxic effects occurred in the strains TA 1535 and TA 98 with and without S9 mix, as well as in the strains TA 1537 and TA 102 without S9 mix all in experiment I and II at higher concentrations. The plates incubated with the test article showed normal background growth up to 5000 ug/plate with and without S9 mix in all strains used. No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with FAT 20202/E; Lanacron Rot S-3G roh feucht at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of significance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.


In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, FAT 20202/E; Lanacron Rot S-3G roh feucht is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.