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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

FAT 20202/E was found to have no mutagenic potential in a bacterial reverse mutation assay. However, FAT 20202/B and FAT 20202/C were shown to be mutagenic and weakly mutagenic in two different bacterial reverse mutation assays esecially in the presence of metabolic activation. This positive effect, however, is considered to be bacterial specific due to the activity of bacterial nitro-reductases, which are highly effective in bacterial strains but not in mammalian cells. Since the molecular structure of Acid Red 407 contains at least two aromatic nitro-groups in total, the presence of DNA-damaging radicals in sufficient amounts is most likely resulting in a false-positive result in the bacterial Ames test. It is well-known for aromatic nitro compounds to be positive in the Ames assay resulting from metabolism by the bacteria-specific enzyme nitro-reductase [Tweats et al., 2012]. However, it has been demonstrated in various publications that this is a bacteria-specific effect and that these Ames positive substances are not mutagenic in mammalian assays. The nitroreductase family comprises a group of flavin mononucleotide (FMN)- or flavin adenine dinucleotide (FAD)-dependent enzymes that are able to metabolize nitroaromatic and nitroheterocyclic derivatives (nitrosubstituted compounds) using the reducing power of nicotinamide adenine dinucleotide (NAD(P)H). These enzymes can be found in bacterial species and, to a lesser extent, in eukaryotes. The nitroreductase proteins play a central role in the activation of nitrocompounds [de Oliveira et al., 2010]. Hence, taking above arguments into account and the negative result from the most recently conducted GLP study with FAT 20202/E, it was concluded that the FAT 20202 is not mutagenic.

Link to relevant study records

Referenceopen allclose all

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
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
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
Target gene:
Histidine auxotrophs
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
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.
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

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
 
 
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.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Study Completion date 10th December, 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
None
Principles of method if other than guideline:
None
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Test Material: FAT 20202/B
Target gene:
Histidine auxotrophic strains of Salmonella typhimurium
Species / strain / cell type:
other: TA 98, TA 100, TA 1535, TA 1537 and TA 1538.
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of liver from rats
Test concentrations with justification for top dose:
500, 1000, 2000, 4000 and 8000 µg/0.1 ml.
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: daunorubicin-HCl
Remarks:
for Strain TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
for Strain TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
for Strain TA 1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
for Strain TA 1537
Details on test system and experimental conditions:
PROCEDURE
The tests were carried out in accordance with the method described by AMES et al.

The bacteria on which the tests were performed were the following histidine-auxotrophic strains of Salmonella typhimurium: TA 98, TA 100, TA 1535, TA 1537 and TA 1538.

The tests were performed with the following concentrations of the trial substance with and without microsomal activation: 25, 75, 225, 675 and 2025 ug/0.1 ml. Additional experiments were carried out on Strains TA 98 and TA 1538 with concentrations of 500, 1000, 2000, 4000 and 8000 ug/0.1 ml. The substance was dissolved in DMSO. DMSO alone was used for the negative controls (the substances and vehicles used for the positive controls are indicated below).
Each Petri dish contained:
1) approx. 20 ml of minimum agar (Agar, Difco Laboratories, Detroit, Michigan, U.S.A., plus salts (Vogel-Bonner Medium E) and glucose),
2) 0.1 ml of the solution of the test substance or the vehicle and 0.1 ml of a bacterial culture (in nutrient broth, Difco Laboratories, Detroit, Michigan, U.S.A., 0.8 % plus 0.5 % NaCl) in 2.0 ml of soft agar. The soft agar was composed of: 100 ml of 0.6 % agar solution with 0.6 % NaCl and 10 ml of a solution of 1-histidine, 0.5 mM (Fluka, Buchs, Switzerland) and +biotin 0.5 mM (Fluka, Buchs, Switzerland). In the experiments in which the substance was metabolically activated, 0.5 ml of an activation mixture was added also (lit.1,2,3). 1 ml activation mixture contains: 0.3 ml S9 fraction of liver from rats induced with Aroclor 1254 (Analabs., Inc., North Haven, Connecticut, U.S.A.) and 0.7 ml of a solution of co-factors.

Positive control experiments were carried out simultaneously with the following substances: 1) for Strain TA 98: daunorubicin-HCl ® (DAUNOBLASTIN , Farmitalia, Montedison Farmaceutica GmbH, Freiburg i.Br., Germany), 5 and 10 ug/0.1 ml phosphate buffer; 2) for Strain TA 100: 4-nitroquinoline-N-oxide (Fluka, Buchs, Switzerland), 0.125 and 0.25 ug/0.1 ml phosphate buffer* 3) for Strain TA 1535: N-Methyl-N'-nitro-N-nitrosoguanidine (Serva, Heidelberg, Germany), 3 and 5 ug/0.1 ml phosphate buffer; 4) for Strain TA 1537: 9(5)aminoacridine hydrochloride monohydrate (Fluka, Buchs, Switzerland), 5 and 10 ug/0.1 ml DMSO. The activation mixture was tested with ® Strain TA 1535 and cyclophosphamide (ENDOXAN-ASTA , Asta-Werke, Bielefeld, Germany), 250 ug/0.1 ml phosphate buffer.

In the experiments without and with the addition of microsomal activation mixture three Petri dishes were prepared per strain and per group (i.e. per concentration or per control group). The plates were incubated for about 48 hours at 37 °C in darkness.
When the colonies had been counted, the arithmetic mean was calculated. The test substance is generally considered to be nonmutagenic if the colony count in relation to the negative control is not doubled at any concentration.
Rationale for test conditions:
None
Evaluation criteria:
None
Statistics:
None
Key result
Species / strain:
other: TA 98, TA 100, TA 1535, TA 1537 and TA 1538
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
None
Remarks on result:
ambiguous mutagenic potential (based on QSAR/QSPR prediction)

Salmonella/Mammalian-Microsome Mutagenicity Test Experiments without microsomal activation Number (arithmetic mean) of colonies of histidine-prototrophic back-mutants:

Strains of S. typhimurium used TA 98 TA 100 TA 1535 TA 1537
FAT 2020202/B CONTROL 21 138 11 4
25 µ / 0.1 ml 23 135 15 4
75 µ / 0.1 ml 18 131 15 5
025 µ / 0.1 ml 23 141 14 6
675 µ / 0.1 ml 21 142 13 8
2025 µ / 0.1 ml 23 152 11 2
Positive Controls        
Daunorubicin-HCl        
Control 21      
5 µ / 0.1 ml 181      
10 µ / 0.1 ml 147      
4-Nitroquinoline-N-oxide        
Control   171    
0.125 µ / 0.1 ml   789    
0.25 µ / 0.1 ml   >1300    
N-methyl-N'-nitro-N-nitrosoguanidine        
       
Control        
3 µ / 0.1 ml     17  
5 µ / 0.1 ml     > 2100  
          > 2400  
9 (5) Aminoacridine hydrochloride        
       
Control       44
50 µ / 0.1 ml       208
100 µ / 0.1 ml       > 1500

Salmonella/Mammalian-Microsome Experiments with microsomal ac Number (arithmetic mean) of co Mutagenicity Test tivation lonies of histidine-prototrophic back-mutants:

Strains of S. typhimurium used TA 98 TA 100 TA 1535 TA 1537
FAT 2020202/B CONTROL 30 128 12 5
25 µ / 0.1 ml 38 132 11 6
75 µ / 0.1 ml 40 133 11 6
025 µ / 0.1 ml 39 133 10 6
675 µ / 0.1 ml 38 132 12 7
2025 µ / 0.1 ml 45 154 12 10
Positive Controls        
Cyclophosphamide        
Control     13  
250 µ / 0.1 ml     415  

Salmonella/Mammalian-Microsome Mutagenicity Test Experiments without microsomal activation Number (arithmetic mean) of colonies of histidine-prototrophic back-mutants:

Strains of S. typhimurium used TA 1538 TA 98 TA 1538
FAT 2020202/B CONTROL 13 20 10
500 µ / 0.1 ml 19 .-. .-.
1000 µ / 0.1 ml 29 36 30
2000 µ / 0.1 ml 15 33 0
4000 µ / 0.1 ml 0 14 0
8000 µ / 0.1 ml 0 5 0
Positive Controls      
Daunorubicin-HCl      
Control   14  
5 µ / 0.1 ml   262  
10 µ / 0.1 ml   283  
2-Nitroflorene      
Control 11   9
5 µ / 0.1 ml 983   > 1000
10 µ / 0.1 ml > 1500   > 1400

Salmonella/Mammalian-Microsome Experiments with microsomal ac Number (arithmetic mean) of co Mutagenici tivation lonies of histidine-prototrophic back-mutants:

Strains of S. typhimurium used TA 1538 TA 98 TA 1538
FAT 2020202/B CONTROL 23 26 20
500 µ / 0.1 ml 23 .-. .-.
1000 µ / 0.1 ml 18 32 25
2000 µ / 0.1 ml 49 37 29
4000 µ / 0.1 ml 72 59 50
8000 µ / 0.1 ml 108 53 61
Positive Controls (microsomal activation)      
Cyclophosphamide      
Control 8    
250 µ / 0.1 ml 502    
Control 8    
250 µ / 0.1 ml 746    
Conclusions:
The metabolites of FAT 20202/B thus exerted a mutagenic action in this test system
Executive summary:

FAT 20202/B was tested for mutagenic effects on histidineauxotrophic mutants of Salmonella typhimurium. The investigations were performed with the following concentrations of the trial substance with and without microsomal activation: 25, 75, 225, 675 and 2025 µg/0.1 ml. In order to confirm the results, additional experiments were performed on strains TA 98 and TA 1538 with concentrations of 500, 1000, 2000, 4000 and 8000 µg/0.1 ml.


These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the numbers of bacteria in the treated and control cultures that have undergone backmutation to histidine-prototrophism. To ensure that mutagenic effects of metabolites of the test substance formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat liver microsomes and co-factors).


In the experiments performed without microsomal activation on strain TA 1538 comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of FAT 20202/B revealed a reduction in the colony count due to a growth-inhibiting effect of the compound at the concentrations of 2000 µg/0.1 ml and above. In the experiments on Strain TA 98, this effect was observed at the concentrations of 4000 and 8000 µg/ 0.1 ml.


In the experiments in which microsomal activation mixture was added, an increase in the number of back-mutant colonies of strain TA 98 was observed at the concentrations of 4000 and 8000 µg/0.1 ml. With strain TA 1538 this effect was observed at concentrations of 2000 µg/0.1 ml and above. The metabolites of FAT 20202/B thus exerted a mutagenic action in this test system

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Experiment Report date: 18th March 1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
None
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only four tester strains tested; no tester strain to detect cross-linking mutagens included
Principles of method if other than guideline:
None
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Test substance: FAT 20202/C
Target gene:
Histidine auxotrophic strains of Salmonella typhimurium
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of liver from rats
Test concentrations with justification for top dose:
500, 1000, 2000, 4000 and 8000 µg/0.1 ml.
Vehicle / solvent:
None
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: daunorubicin- HCl
Remarks:
for strain TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
for strain TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
for strain TA 1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
for strain TA 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
for strain TA 1538
Details on test system and experimental conditions:
PROCEDURE
The tests were carried out in accordance with the method described by AMES et al. The bacteria on which the tests were performed were the following histidine-auxotrophic strains of Salmonella typhimurium: TA 98, TA 100, TA 1535, TA 1537 and TA 1538. The tests were performed with the following concentrations of the trial substance without and with microsomal activation: 25, 75, 225, 675 and 2025 ug/0.1 ml. Additional experiments were performed with the concentrations of 500, 1000, 2000, 4000 and 8000 ug/0.1 ml. The substance was dissolved in phosphate buffer. Phosphate buffer alone was used for the negative controls (the substances and vehicles used for the positive controls are indicated below).
Each Petri dish contained:

1) approx. 20 ml of minimum agar (Agar, Difco Laboratories, Detroit, Michigan, U.S.A., plus salts (Vogel-Bonner Medium E) and glucose),
2) 0.1 ml of the solution of the test substance or the vehicle and 0.1 ml of a bacterial culture (in nutrient broth, Difco Laboratories, Detroit, Michigan, U.S.A., 0.8 % plus 0.5 % NaCl) in 2.0 ml of soft agar. The soft agar was composed of: 100 ml of 0.6 % agar solution with 0.6 % NaCl and 10 ml of a solution of 1-histidine, 0.5 mM (Fluka, Buchs, Switzerland) and +biotin 0.5 mM (Fluka, Buchs, Switzerland). In the experiments in which the substance was metabolically activated, 0.5 ml of an activation mixture was added also (lit.1,2,3). 1 ml activation mixture contained: 0.3 ml S9 fraction of liver from rats induced with Aroclor 1254 (Analabs., Inc., North Haven, Connecticut, U.S.A.) and 0.7 ml of a solution of co-factors.

Positive control experiments were carried out simultaneously with the following substances:
1) for strain TA 98: daunorubicin- HCl (DAUNOBLASTIN , Farmitalia, Montedison Farmaceutica GmbH, Freiburg i.Br., Germany), 5 and 10 ug/0.1 ml phosphate buffer;
2) for strain TA 100: 4-nitroquinoline-N-oxide (Fluka, Buchs, Switzerland), 0.125 and 0.25 p.q/0.1 ml phosphate buffer;
3) for strain TA 1535: N-methyl-N'-nitro-N-nitrosoguanidine (Serva, Heidelberg, Germany), 3 and 5
µg/0.1 ml phosphate buffer;
4) for strain TA 1537: 9(5)aminoacridine hydrochloride monohydrate (Fluka, Buchs, Switzerland), 50 and 100 pg/0.1 ml DMSO;
5) for strain TA 1538: 2-nitrofluorene (Fluka, Buchs, Switzerland), 5 and 10 ug/0.1 ml DMSO. The activation mixture was ® tested with strain TA 1535 and cyclophosphamide (ENDOXAN-ASTA , Asta-Werke, Bielefeld, Germany), 250 µg/0.1 ml phosphate buffer.

In the experiments without and with the addition of microsomal activation mixture three Petri dishes were prepared per strain and per group (i.e. per concentration or per control group). The plates were incubated for about 48 hours at 37 °C in darkness. When the colonies had been counted, the arithmetic mean was calculated. The test substance is generally considered to be nonmutagenic if the colony count in relation to the negative control is not doubled at any concentration.
Rationale for test conditions:
None
Evaluation criteria:
None
Statistics:
None
Key result
Species / strain:
other: TA 98, TA 100, TA 1535 and TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
None

SALMONELLA/MAMMALIAN -MICROSOME MUTAGENICITY TEST EXPERIMENTS WITHOUT MICROSOMAL ACTIVATION NUMBER OF BACK-MUTANT COLONIES PER PLATE (ARITHMETIC MEAN):

Strains of S. typhimurium used TA 98 TA 100 TA 1535 TA 1537
CONTROL 7 142 9 7
25 µ / 0.1 ml 2 141 14 5
75 µ / 0.1 ml 14 157 7 6
225 µ / 0.1 ml 12 141 5 9
675 µ / 0.1 ml 9 166 1 0
2025 µ / 0.1 ml 0 162 2 0
Positive Controls        
Daunorubicin-HCl        
Control 16      
5 µ / 0.1 ml 70      
10 µ / 0.1 ml 99      
4-Nitroquinoline-N-oxide        
Control   139    
0.125 µ / 0.1 ml   1154    
0.25 µ / 0.1 ml   1033    
N-methyl-N'-nitro-N-nitrosoguanidine        
       
Control        
3 µ / 0.1 ml     9  
5 µ / 0.1 ml     903  
          2122  
9 (5) Aminoacridine hydrochloride        
       
Control       5
50 µ / 0.1 ml       62
100 µ / 0.1 ml       357

SALMONELLA/MAMMALI AN-MICROSOME MUTAGENICITY TEST EXPERIMENTS WITH MICROSOMAL ACTIVATION NUMBER OF BACK-MUTANT COLONIES PER PLATE (ARITHMETIC MEAN):

Strains of S. typhimurium used TA 98 TA 100 TA 1535 TA 1537
CONTROL 39 129 10 8
25 µ / 0.1 ml 44 121 8 8
75 µ / 0.1 ml 47 122 10 4
025 µ / 0.1 ml 29 144 47 6
675 µ / 0.1 ml 30 160 10 6
2025 µ / 0.1 ml 65 164 7 9
Positive Controls        
Cyclophosphamide        
Control     9  
250 µ / 0.1 ml     460  

SALMONELLA/MAMMALIAN-MICROSOME MUTAGENICITY TEST EXPERIMENTS WITHOUT MICROSOMAL ACTIVATION NUMBER OF BACK-MUTANT COLONIES PER PLATE (ARITHMETIC MEAN):

Strains of S. typhimurium used TA 98 TA 100 TA 1535 TA 1537 TA 1538
CONTROL 26 198 19 5 15
500 µ / 0.1 ml 36 165 13 8 40
1000 µ / 0.1 ml 22 173 4 7 24
2000 µ / 0.1 ml 0 204 2 1 2
4000 µ / 0.1 ml 4 216 1 1 1
8000 µ / 0.1 ml 0 226 0 0 0
Positive Controls          
Daunorubicin-HCl          
Control 27        
5 µ / 0.1 ml 418        
10 µ / 0.1 ml 644        
4-Nitroquinoline-N-oxide          
Control   213      
0.125 µ / 0.1 ml   714      
0.25 µ / 0.1 ml   1239      
N-methyl-N'-nitro-N-nitrosoguanidine          
         
Control     19    
3 µ / 0.1 ml     1305    
5 µ / 0.1 ml     2517    
9 (5) Aminoacridine hydrochloride          
         
Control       6  
50 µ / 0.1 ml       38  
100 µ / 0.1 ml       560  
2-Nitroflorene          
Control         15
5 µ / 0.1 ml         967
10 µ / 0.1 ml         1405

SALMONELLA/MAMMALIAN-MICROSOME MUTAGENICITY TEST EXPERIMENTS WITH MICROSOMAL ACTIVATION NUMBER OF BACK-MUTANT COLONIES PER PLATE (ARITHMETIC MEAN):

Strains of S. typhimurium used TA 98 TA 100 TA 1535 TA 1537 TA 1538
CONTROL 56 155 12 4 22
500 µ / 0.1 ml 48 157 12 2 28
1000 µ / 0.1 ml 52 165 10 10 26
2000 µ / 0.1 ml 49 131 10 13 41
4000 µ / 0.1 ml 70 164 14 11 80
8000 µ / 0.1 ml 11 190 0 7 51
Positive Controls          
Cycophosphamide          
Control     15    
250 µ / 0.1 ml     494    
Conclusions:
FAT 20202/C and its metabolites formed as a result of microsomal activation, displayed an albeit very weak mutagenic action in this test system.
Executive summary:

FAT 20202/C was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium. The investigations were performed with strains TA 98, TA 100, TA 1535 and TA 1537 with the following concentrations of the trial substance without and with microsomal activation: 25, 75, 225, 675 and 2025 µg/0.1 ml. In order to confirm the results, additional experiments were performed with all strains including TA 1538 with the concentrations of 500, 1000, 2000, 4000 and 8000 µg/0.1 ml. These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the numbers of bacteria in the treated and control cultures that have undergone back-mutation to histidine-prototrophism. To ensure that mutagenic effects of metabolites of the test substances formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat liver microsomes and co-factors). In the experiments without microsomal activation on strains TA 98 and TA 1538, treatment with FAT 20202/C led to a slight increase in the number of back-mutant colonies at the concentrations of 75 and 500 µg/0.1 ml, respectively. This effect occurred also in the experiments with microsomal activation at the concentrations of 2025 µg/0.1 ml (strain TA 98) and 2000 to 8000 µg/0.1 ml (strain TA 1538). FAT 20202/C and its metabolites formed as a result of microsomal activation, thus, displayed an albeit very weak mutagenic action in this test system.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

None

Additional information

Three bacterial reverse mutation assays are available for assessment of mutagenicity potential of the substance FAT 20202. In a key study, 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; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 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 is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay. 


In a supporting study conducted in 1980, FAT 20202/B was tested for mutagenic effects on histidineauxotrophic mutants of Salmonella typhimurium. The investigations were performed with the following concentrations of the trial substance with and without microsomal activation: 25, 75, 225, 675 and 2025 µg/0.1 ml. In order to confirm the results, additional experiments were performed on Strains TA 98 and TA 1538 with concentrations of 500, 1000, 2000, 4000 and 8000 µg/0.1 ml. These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the numbers of bacteria in the treated and control cultures that have undergone back mutation to histidine-prototrophism. To ensure that mutagenic effects of metabolites of the test substance formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat liver microsomes and co-factors). In the experiments performed without microsomal activation on strain TA 1538 comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of FAT 20202/B revealed a reduction in the colony count due to a growth-inhibiting effect of the compound at the concentrations of 2000 µg/0.1 ml and above. In the experiments conducted on strain TA 98, this effect was observed at the concentrations of 4000 and 8000 µg/ 0.1 ml. In the experiments in which microsomal activation mixture was added, an increase in the number of back-mutant colonies of strain TA 98 was observed at the concentrations of 4000 and 8000 µg/0.1 ml. With strain TA 1538 this effect was observed at concentrations of 2000 µg/0.1 ml and above. Hence, it was concluded that the metabolites of FAT 20202/B exerted a mutagenic action in this test system.


 


In another supporting study (1982), FAT 20202/C was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium. The investigations were performed with strains TA 98, TA 100, TA 1535 and TA 1537 with the following concentrations of the trial substance without and with microsomal activation: 25, 75, 225, 675 and 2025 µg/ 0.1 ml. In order to confirm the results, additional experiments were performed with all strains including TA 1538 with the concentrations of 500, 1000, 2000, 4000 and 8000 µg/0.1 ml. These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the numbers of bacteria in the treated and control cultures that have undergone back-mutation to histidine-prototrophism. To ensure that mutagenic effects of metabolites of the test substances formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat liver microsomes and co-factors). In the experiments without microsomal activation on strains TA 98 and TA 1538, treatment with FAT 20202/C led to a slight increase in the number of back-mutant colonies at the concentrations of 75 and 500 µg/0.1 ml respectively. This effect occurred also in the experiments with microsomal activation at the concentrations of 2025 µg/0.1 ml (strain TA 98) and 2000 to 8000 µg/0.1 ml (strain TA 1538). Hence, it was concluded that FAT 20202/C and its metabolites formed as a result of microsomal activation displayed an albeit very weak mutagenic action in this test system.


 


In the study conducted with FAT 20202/B, the assay was clearly negative without metabolic activation, however a slight increase in the number of back-mutant colonies of strain TA 98 and strain TA 1538 was observed in the presence of metabolic activation. In the second study conducted with FAT 20202/C, a slight increase in the number of back-mutant colonies at the concentrations of 75 and 500 µg/0.1 ml, respectively, which are the low doses, was seen, while no such effect was observed at higher doses. This can be considered as a background noise for the assay. This effect also occurred in the experiments with microsomal activation at the concentrations of 2025 µg/0.1 ml (strain TA 98) and 2000 to 8000 µg/0.1 ml (strain TA 1538).


 


This positive effect in the bacterial reverse mutation assay in the presence of metabolic activation is considered to be bacterial specific due to the activity of bacterial nitro-reductases, which are highly effective in bacterial strains but not in mammalian cells. Since the molecular structure of Acid red 407 contains at least two aromatic nitro-groups in total, the presence of DNA-damaging radicals in sufficient amounts is most likely resulting in a false-positive result in the bacterial Ames test.


 


 It is well-known for aromatic nitro compounds to be positive in the Ames assay resulting from metabolism by the bacteria-specific enzyme nitro-reductase [Tweats et al., 2012]. However, it has been demonstrated in various publications that this is a bacteria-specific effect and that these Ames positive substances are not mutagenic in mammalian assays. The nitroreductase family comprises a group of flavin mononucleotide (FMN)- or flavin adenine dinucleotide (FAD)-dependent enzymes that are able to metabolize nitroaromatic and nitroheterocyclic derivatives (nitrosubstituted compounds) using the reducing power of nicotinamide adenine dinucleotide (NAD(P)H). These enzymes can be found in bacterial species and, to a lesser extent, in eukaryotes. The nitroreductase proteins play a central role in the activation of nitrocompounds [de Oliveira et al., 2010]. Hence, taking into account above argument and a negative result from the most recently conducted GLP study with FAT 20202/E, it was concluded that the FAT 20202 is not mutagenic.

Justification for classification or non-classification

Based on the above information, the substance does not warrant classification for genotoxicity as per the CLP (Regulation 1272/2008) criteria.