Registration Dossier
Registration Dossier
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Reaction mass of tetrasodium 5-{[4-chloro-6-(4-{[2-(sulfonatooxy)ethyl]sulfonyl}anilino)-1,3,5-triazin-2-yl]amino}-4-hydroxy-3-[(4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl) diazenyl]naphthalene-2,7-disulfonate and trisodium 5-({4-chloro-6-[4-(vinylsulfonyl) anilino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-2,7-disulfonate
EC number: 701-360-8 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The test substance did not show genotoxic properties in in-vitro tests
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 11 October to 20 October 1989
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 84/449/EWG, B.14, Prival Modification
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- TA98 hisD3052 Frameshift
TA100 hisG46 Base pair substitution
TA1535 hisG46 Base pair substitution
TA1537 hisC3076 Frameshift
TA1538 hisD3052 Frameshift - Species / strain / cell type:
- S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
- Additional strain / cell type characteristics:
- other: histidine dependent
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-Mix from rat and hamster liver
- Test concentrations with justification for top dose:
- Preliminary test: 4 to 10000 µg/plate
Main test: 4 to 5000 µg/plate - Vehicle / solvent:
- At the day of the experiment the test substance was dissolved in Aqua bidest at appropriate concentrations. Two independent experiments were performed for each protocol (Ames, Prival).
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- aqua bidest
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- w/o S9 Migrated to IUCLID6: TA100, TA1535
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- w/o S9 Migrated to IUCLID6: TA1537
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- Remarks:
- w/o S9 Migrated to IUCLID6: TA1538, TA98
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- with rat S9 10%: TA98, TA100, TA1535, TA1537, TA1538; with rat S9 30%: TA98, TA100, TA1535, TA1537
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- with rat S9 10%: TA98, TA100, TA1535, TA1537, TA1538; with rat S9 30%: TA98, TA100, TA1535, TA1537
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- congo red
- Remarks:
- with hamster S9 30%: TA98
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: benzidine
- Remarks:
- with hamster S9 30%: TA98
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- with hamster S9 30%: TA100, TA1535, TA1537
- Details on test system and experimental conditions:
- Preparation and storage of a liver homogenate fraction ("S-9")
Liver preparations were performed from liver of Aroclor induced Sprague Dawley rats and from non pretreated Syrien hamsters. Male Sprague Dawley rats (200 -300 g) receive a single intraperitoneal injection of Aroclor 1254 (500 mg/kg bodyweight) 5 days before sacrifice. Preparation is performed at 0 to 4 °C using cold sterile solution and glassware. The livers from at least 5-6 Sprague Dawley rats or from 5-6 male Syrian golden hamsters (7-8 weeks old) are removed and pooled, washed in 150 mM KCl (approximately 1 ml/g wet livers). The washed livers are cut into small pieces and homogenized in three volumes of KC1. The homogenate is centrifuged at 9000 g for 10 minutes. The supernatant is the S-9 fraction. It is divided into small portions, rapidly frozen and stored at -80 °C for not longer than three months.
Preparation of S-9 Mix
Sufficient S-9 fraction is thawed immediately before each test at room tempera¬ture. One or three volumes of S-9 fraction is mixed with nine or seven volumes of the S-9 cofactor solution and kept on ice until used. This preparation is termed S-9 Mix. The concentrations of the different compounds in the S-9 Mix of the rat liver are:
8 mM MgCl2
33 mM KC1
5 mM glucose-6-phosphate
4 mM NADP+
100 mM phosphate buffer pH 7.4
According to the modification proposed by Prival (5) using 30 minutes preincubation in the presence of 30 % Syrian golden hamster S-9 Mix. The S-9 Mix consists of:
8 mM MgCl2
33 mM KCI
20 mM glucose-6-phosphate
2.8 units/ml glucose-6-phosphate dehydrogenase
4 mM NAPD+
2 mM NADH
2 mM FMN (Riboflavin-5’-phosphate-Na-salz)
100 mM phosphate buffer pH 7.4
Bacteria
Bacteria are grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No 2 /liter) at 37 °C. The suitable amount of bacteria in the cell suspension is checked by nephelometry. For inoculation, stock cultures which are stored at -80 °C, are used. The compound is tested with the strains Salmonella typhimurium TA 100, TA 1535, TA 1537, TA 1538 and TA 98.
Toxicity experiments and dose range finding
Preliminary toxicity tests were performed with five or four tester strains using three plates per dose to get information on mutagenicity and toxicity for calculation of an appropriate dose range. A reduced rate of spontaneously occuring colonies as well as visible thinning of the bacterial lawn were used as indicator for toxicity. Thinning of the bacterial lawn was controlled microscopically. In combination with the main experiments, toxicity testing was performed as follows: 0.1 ml of the different dilutions of the test compound were thoroughly mixed with 0.1 ml of 10-6 dilution of the overnight culture of TA 100 and plated with histidine and biotin rich top agar (3 plates per dose). The solvent control is compared with the number of colonies per plate in the presence of the test compound. Results are given as a ratio of these values (= surviving fraction).
Mutagenicity test
Two independent experiments which each of the two protocols (Ames, Prival) were performed.
a) - with 10 % rat liver S-9 Mix or buffer and the strains TA 100, TA 1535, TA 1537, TA 1538 and TA 98 - with 30 % rat liver S-9 Mix and the stains TA 100, TA 1535, TA 1537 and TA 98
Top agar is prepared for the Salmonella strains by mixing 100 ml agar (0.6 % agar, 0.6 % NaCl) with 10 ml of a 0.5 mM histidine-biotin solution. The follow¬ing ingredients are added (in order) to 2 ml of molten top agar at 45 °C:
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.1 ml test compound solution
0.5 ml 10 % or 30 % rat liver S-9 Mix or buffer
After mixing, the liquid is poured into a petridish with minimal agar (1.2 % agar, Vogel-Bonner E medium with 2 % glucose). After incubation for 48 to 72 hours at 37 °C in the dark, colonies (his+ revertants) are counted.
b) with 30 % Syrian golden hamster S-9 Mix and preincubation
0.1 ml test solution, 0.1 ml bacterial suspension and 0.5 ml S-9 Mix are incubated at 30 °C for the duration of 30 minutes. Subsequently, 2 ml of soft agar which consists of 100 ml agar (0.6 % agar + 0.6 % NaCl) and 10 ml amino-acid solution (minimal amino-acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) is added. After mixing, the samples are poured on to the Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds. After incubation for 48 to 72 hours at 37 °C in the dark, colonies (his+ revertants) are counted.
Positive controls
Positive control plates were included for each strain. The following substances were used as positive controls.
a) without metabolic activation:
Na-azide: TA 100, TA 1535
9-Aminoacridine: TA 1537
2-Nitrofluorene: TA 1538, TA 98
b) with rat liver S-9 Mix (10 %):
Benzo[a]pyrene: TA 98, TA 100, TA 1535, TA 1537, TA 1538
2-Aminoanthracene: TA 98, TA 100, TA 1535, TA 1537, TA 1538
c) with rat liver S-9 Mix (30 %):
Benzo[a]pyrene: TA 98, TA 100, TA 1535, TA 1537
2-Aminoanthracene: TA 98, TA 100, TA 1535, TA 1537
d) with hamster liver S-9 Mix (30 %):
2-Aminoanthracene: TA 100, TA 1535, TA 1537
Benzidine: TA 98
Congored: TA 98 - Evaluation criteria:
- No data
- Statistics:
- No data
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- 10% rat S9
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- up to 10000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- 10% rat S9, 30% rat S9, 30% hamster S9
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- up to 10000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- 10% rat S9
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- up to 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- 10% rat S9, 30% rat S9, 30% hamster S9
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- up to 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Reaktiv-Rot F-66 813 FW was tested for mutagenicity with Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 in the absence and presence of a metabolic activation systems. S-9 Mix from Sprague Dawley rats (10 % or 30 %) and from Syrien golden hamsters (30 %) were used. The number of colonies per plate with each strain as well as mean values of 3 plates, corrected to the next whole number are given.
Sterility checks and control plates
Sterility of S-9 Mix and the test compound were indicated by the absence of contamination on the test material and S-9 Mix sterility check plates. Control plates (background control and positive controls) gave the expected number of colonies.
Toxicity test
The test compound was tested at doses of 4 to 10000 microgram/plate and proved to be not toxic to the bacterial strains. For mutagenicity testing 5000 microgram/plate was chosen as the highest dose in the main experiments.
Mutagenicity test with Reaktiv-Rot F-66 813 FW
Ames-Test:
The test compound did not cause a significant increase in the number of revertant colonies with any of the tester strains either in the absence or in the presence of rat S-9 Mix (10 %). No dose dependent effect was obtained.
Prival-Test:
In the presence of rat liver S-9 Mix (30 %) and hamster liver S-9 Mix (30 %) using the preincubation method according to Prival the test compound did not show any relevant increases in the number of revertant colonies under the experimental conditions described. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'. Remarks: preliminary test
- Conclusions:
- Interpretation of results (migrated information):
negative
It can be stated that Reaktiv-Rot F-66 813 FW is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival. - Executive summary:
This study followed the procedures indicated by the following internationally accepted guidelines and recommendations:OECD Guidelines for testing of chemicals 471Genetic Toxicology : Salmonella typhimurium, Reverse Mutation Assay, Adopted : May 26th, 1983andEEC Directive 79/831 Annex V, 4.3.1. This study was conducted in compliance with the principles of good laboratory practice (GLP).
Reaktiv-Rot F-66 813 FW was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 1538 and TA 98 of Salmonella typhimurium.
The mutagenicity studies were conducted in the standard plate test (Ames Test) and in a modified preincubation test (Prival Test). The studies were performed in the absence and in the presence of a metabolizing system derived from rat or hamster liver homogenate. A dose range of 6 different doses from 4 microgram/ plate to 5000 microgram/plate was used.
Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.
Toxicity: The test compound proved to be not toxic to the bacterial strains. 5 000 microgram/plate was chosen as top dose level for the mutagenicity study.
Ames Test:
Mutagenicity: In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of rat liver activation system (10 %), treatment of the cells with Reaktiv-Rot F-66 813 FW did not result in relevant increases in the number of revertant colonies.
Prival Test:
In the presence of rat liver S-9 Mix (30 %) and hamster liver S-9 Mix (30 %) using the preincubation method according to Prival Reaktiv-Rot F-66 813 FW did not induce a significant increase in the number of revertant colonies, with any of the tester strains.
Summarizing, it can be stated that Reaktiv-Rot F-66 813 FW is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 18 July 2003 to 28 August 2003
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- 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)
- Version / remarks:
- incorporating Privall Mitchell preincubation test
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- JSCL TG III.1 Gene mutation test with bacteria
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Salmonella typhimurium:
TA98 hisD3052 Frameshift
TA100 hisG46 Base pair substitution
TA1535 hisG46 Base pair substitution
TA1537 hisC3076 Frameshift
Escherichia coli:
WP2uvrA trpE Base pair substitution - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- other: histidine dependent
- Species / strain / cell type:
- E. coli WP2 uvr A pKM 101
- Additional strain / cell type characteristics:
- other: tryptophan dependent
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-Mix from rat and hamster liver
- Test concentrations with justification for top dose:
- 50, 160, 500, 1600, 5000 µg/plate
- Vehicle / solvent:
- deionised water
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- w/o S9 Migrated to IUCLID6: TA100, TA1535
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- w/o S9 Migrated to IUCLID6: TA1537
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- Remarks:
- w/o S9 Migrated to IUCLID6: TA98
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- w/o S9 Migrated to IUCLID6: WP2uvrA
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- with rat S9 10%: TA98, TA100, TA1535, TA1537, WP2uvrA
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- congo red
- Remarks:
- with hamster S9 30% Migrated to IUCLID6: TA98
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- with hamster S9 30%: TA100, TA1535, TA1537, WP2uvrA
- Details on test system and experimental conditions:
- Test groups
plate incorporation test:
with metabolic activation(10 % rat liver): 50,160, 500, 1600 and 5000 μg/plate b:
without metabolic activation: 50, 160, 500, 1600 and 5000 μg/plate
preincubation test:
with metabolic activation (30 % hamster liver): 50, 160, 500, 1600 and 5000 μg/plate
without metabolic activation: 50, 160, 500, 1600 and 5000 μg/plate
Control groups
negative controls:
a: untreated controls
b: solvent controls (0 μg/plate)
positive controls:
without metabolic activation: sodium-azide for strain TA 100 and TA 1535, 9-aminoacridine for strain TA 1537, 2-nitrofluorene for strain TA 98, 4-nitroquinoline-N-oxide for strain WP2uvrA
with metabolic activation (10 % rat liver): 2-aminoanthracene for all strains
with metabolic activation (30 % syrian golden hamster liver): 2-aminoanthracene for strain TA 100, TA 1535, TA 1537 and WP2uvrA, congo red for strain TA 98
Formulation of test compound: dissolved in deionised water at appropriate concentrations immediately before use.
Formulation of reference compounds
Sodium-azide dissolved in deionised water final concentration: 1.0μg/plate for strain TA 1535, 2.0μg/plate for strain TA 100
9-aminoacridine dissolved in DMSO final concentration: 50.0μg/plate for strain TA 1537
2-nitrofluorene dissolved in DMSO final concentration: 2.5μg/plate for strain AT 98
4-nitroquinoline-N-oxide dissolved in DMSO final concentrations: 2.0μg/plate (plate inc.), 0.5μg/plate (preinc.) for strain WP2uvrA
2-aminoanthracene dissolved in DMSO final concentrations: (10% (v/v) rat liver S9-mix): 1.5μg/plate for strains TA 98, TA 100, TA 1535 and TA 1537, 20.0μg/plate for strain WP2uvrA
2-aminoanthracene dissolved in DMSO final concentrations (30% (v/v) hamster liver S9-mix): 1.0μg/plate for strain TA 100, TA 1535 and TA 1537, 30.0μg/plate for strain WP2uvrA
Congo red dissolved in deionised water final concentration: 250μg/plate for strain TA98
The frozen stock solutions of each compounds were diluted progressively up to the final concentration on the day of treatment.
Source of bacteria: stock cultures in the bank of “Genetic Toxicology”, Aventis Pharma Germany, ProTox prepared from the original bacteria strains.
Test organism: Salmonella typhimurium strains – TA 98 hisD3052 rfa uvrB +R, TA 100 hisG46 rfa uvrB +R, TA 1535 hisG46 rfa uvrB, TA 1537 hisC3076 rfa uvrB and Escherichia coli WP2uvrA pKM101
Experimental conditions in vitro: approx. 37°C in an incubator.
Preparation and storage of a liver homogenate fraction (S9)
The S9 fraction of Spraque Dawley rat liver induced with Aroclor 1254 was obtained by Molecular Toxicology, Inc., 157 Industrial Park Dr. Boone, NC 28607, (828) 264-9099. The protein content for every batch was guaranteed by a Quality Control & Production Certificate by the supplier. Also for every batch of S9 an independent validation was performed in the laboratory with a minimum of two different mutagens, e.g. 2-aminoanthracene and benzo(a)pyrene, to confirm metabolic activation by microsomal enzymes.
The S9 fraction of Syrian golden hamster liver was prepared by the department conducting the study according to Prival et. al (1982). Male Syrian golden hamsters (7-8 weeks old), were supplied by Harlan Winkelmann, Gartenstrasse 27, 33178 Borchen, Germany. Liver preparations were performed from the liver of non pretreated Syrian hamsters. The livers were removed from 10 male Syrian hamsters (7-8 weeks old) using cold sterile solutions at approx. 0 to 4 °C and glassware, and were then pooled and washed in approx. 150 mM KC1 (approximately 1 ml/g wet liver). The washed livers were cut into small pieces and homogenized in three volumes of KC1. The homogenate was centrifuged at approx. 9000g for 10 minutes. The supernatant was the S9 fraction. This was divided into small portions, rapidly frozen and stored at approx. - 80 °C. The protein content was determined for every batch. Also for every batch of S9 an independent validation was performed with a minimum of two different mutagens, e.g. 2-aminoanthracene and congo red, to confirm metabolic activation by microsomal enzymes.
Preparation of S9-mix
Sufficient S9 fraction was thawed at room temperature immediately before each test. One volume of Moltox. S9 fraction (batch no. 1530 for the plate incorporation test, protein concentration 36.1 g/l) was mixed with 9 volumes of the S9 cofactor solution, which was kept on ice until used. This preparation is termed S9-mix. The concentrations of the different compounds in the rat liver S9-mix were:
8 mM MgCl2
33 mM KC1
5 mM glucose-6-phosphate
4mM NADP
100 mM phosphate buffer pH 7.4
According to the modification proposed by Prival (8) the test substance and the tester strains were
preincubated for 20 to 30 minutes with 30 % (v/v) Syrian golden hamster S9-mix.
Three volumes of S9 fraction (batch no. 2002/1 for the preincubation, protein concentration
45 g/l) were mixed with 7 volumes of the S9 cofactor solution.
This preparation is termed S9-mix. The hamster liver S9-mix consists of:
8 mM MgCl2
33 mM KC1
20 mM glucose-6-phosphate
2.8 units/ml glucose-6-phosphate dehydrogenase
4mM NADP+
2 mM NADH
2 mM FMN (Riboflavine-5’-phosphate-sodium-salt)
100 mM phosphate buffer pH 7.4
Bacteria
The strains of Salmonella typhimurium were obtained from Professor B.N. Ames, University of California, U.S.A. The strain E. coli was obtained from E.coli Genetic Stock Center, Yale University, New Haven, U.S.A.
Bacteria were grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No. 2 /liter) at approx. 37 °C. The amount of bacteria in the cell suspension was checked by nephelometry. Inoculation was performed with stock cultures which had been stored in liquid nitrogen. Each new stock of the different bacterial strains was checked with regard to the respective biotin and histidine requirements, membrane permeability, ampicillin resistance, tetracyclin resistance, crystal violet sensitivity, UV resistance and response to diagnostic mutagens.
ASSAY PROCEDURE
An independent mutation test was performed using the plate incorporation method. When results were negative or equivocal, a second test was conducted. This included a preincubation step if the first test was clearly negative. Preincubation involved incubating the test substance, S9-mix and bacteria for a short period before pouring this mixture onto plates of minimal agar.
Each test was performed in both the presence and absence of S9-mix using all bacterial tester strains and a range of concentrations of the test substance. Positive and negative controls as well as solvent controls were included in each test. Triplicate plates were used. The highest concentration in the first mutation experiment was 50 mg/ml of the test substance in the chosen solvent, which provided a final concentration of 5000μg/plate. Further dilutions of 1600, 500, 160 and 50μg/plate were also used. Dose levels used in the second experiment were based on findings, including toxicity, in the first experiment. Toxicity was assessed after microscopic thinning of the bacterial lawn and/or reduction of the number of spontaneously occurring mutants compared to the corresponding solvent control value.
In both tests top agar was prepared which, for the Salmonella strains, contained 100 ml agar (0.6 % (w/v) agar, 0.5 % (w/v) NaCl) with 10 ml of a 0.5 mM histidine-biotin solution. For E. coli histidine was replaced by tryptophan (2.5 ml, 2.0 mM). The following ingredients were added (in the following order) to 2 ml of molten top agar at approx. 48 °C:
0.5 ml S9-mix (if required) or buffer
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.1 ml test compound solution (dissolved in deionised water)
In the second mutagenicity test if appropriate these top-agar ingredients were preincubated by shaking for approximately 20 to 30 minutes at approx. 30 °C.
After mixing, and preincubation if appropriate, the liquid was poured into a petri dish containing a 25 ml layer of minimal agar (1.5 % (w/v) agar, Vogel-Bonner E medium with 2 % (w/v) glucose).
After incubation for approximately 48 hours at approx. 37 °C in the dark, colonies (his+ or trp+ revertants) were counted by hand or by a suitable automatic colony counter. The counter was calibrated for each test by reading a test pattern plate to verify the manufacturer's requirements for sensitivity. - Evaluation criteria:
- Criteria for a valid assay
The assay is considered valid if the following criteria are met:
the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
the positive controls induce increases in the mutation frequency which are significant and within the laboratory's normal range
Criteria for a positive response
A test compound is classified as mutagenic if it has either of the following effects:
it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn
If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system. - Statistics:
- No data
- Species / strain:
- E. coli WP2 uvr A pKM 101
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- 10% rat S9
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- up to 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- 10% rat S9
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- up to 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A pKM 101
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- 30% hamster S9
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- up to 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- 30% hamster S9
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- up to 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- STERILITY CHECKS AND CONTROL PLATES
Sterility of S9-mix and the test compound were indicated by the absence of contamination on the test material and S9-mix sterility check plates. Control plates (background control and positive controls) gave the expected number of colonies, i.e. values were within the laboratory's historical control range.
The number of revertant colonies of the solvent controls with the strain TA 100 in the absence of S9-mix in the plate incorporation test was below the historical control data range, but the criteria for the negative response were fulfilled.
The number of revertant colonies of the positive controls with the strains TA 1535, TA 98 and WP2 uvrA in the presence of S9-mix in the preincubation test was above the historical control data range, but the criteria for the positive response were fulfilled. Also in the preincubation test the number of revertant colonies with the strain TA 1537 was above the historical control data range, but the criteria for the negative/positive response were fulfilled.
SOLUBILITY AND TOXICITY
Reaktiv Rot F66813 was dissolved in deionized water and a stock solution of 50 mg/ml was prepared for the highest concentration, which provided a final concentration of 5000μg/plate. Further dilutions of 1600, 500, 160 and 50μg/plate were used in all experiments.
Reaktiv Rot F66813 did not precipitate on the plates up to the highest investigated dose of 5000μg/plate.
Reaktiv Rot F66813 proved to be not toxic to the bacterial strains.
MUTAGENICITY
The number of colonies per plate with each strain as well as mean values of 3 plates were given.
Plate incorporation test:
The test compound did not cause a significant increase in the number of revertant colonies at any dose level with any of the tester strains either in the absence or presence of rat liver S9-mix in either mutation test. No dose-dependent effect was obtained.
Preincubation test:
In the presence of hamster liver S9-mix (30 % (v/v)) using the preincubation method according to Prival the test compound did not cause a significant increase in the number of revertant colonies under the experimental conditions described. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'. Remarks: plate incorporation test
- Conclusions:
- Interpretation of results (migrated information):
negative
The results lead to the conclusion that Reaktiv Rot F66813 is not mutagenic in the absence and presence of metabolic activation using the standard Ames Test procedure (plate incorporation test) and the preincubation test as described. - Executive summary:
The present study was conducted in compliance with OECD Guideline For Testing Of Chemicals, 471 Bacterial Reverse Mutation Test Adopted: 21-July-1997andU.S. EPA: OPPTS 870.5100 Health Effects Test Guidelines Bacterial Reverse Mutation Test, Aug-1998andEC Directive 2000/32/EC, L 136, Annex 4D, B.13/B.14andJapanese Substance Control Law (JSCL) Test Guideline III.l Gene Mutation Test with bacteria.The study is based on the Principles ofGood Laboratory Practice (GLP).
Reaktiv Rot F66813 (Batch No. Op. 1/98) was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 98 of Salmonella typhimurium and with Escherichia coli WP2uvrA. Two independent mutagenicity studies were conducted, one as the standard plate test with the plate incorporation method and the other as a modified preincubation test (Prival test). The studies were performed in the absence and in the presence of a metabolizing system derived from a rat liver homogenate or a hamster liver homogenate.
For all studies, the compound was dissolved in deionised water, and each bacterial strain was exposed to 5 dose levels. Doses for both studies ranged from 50 to 5000μg/plate.
Reaktiv Rot F66813 did not precipitate on the plates up to the highest investigated dose of 5000μg/plate.
Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control. All positive controls gave the expected increase in the number of revertant colonies.
The number of revertant colonies of the solvent controls with the strain TA 100 in the absence of S9-mix in the plate incorporation test was below the historical control data range, but the criteria for the negative response were fulfilled.
The number of revertant colonies of the positive controls with the strains TA 1535, TA 98 and WP2 uvrA in the presence of S9-mix in the preincubation test was above the historical control data range, but the criteria for the positive response were fulfilled. Also in the preincubation test the number of revertant colonies with the strain TA 1537 was above the historical control data range, but the criteria for the negative/positive response were fulfilled.
Toxicity: In both studies toxicity was not observed either with or without metabolic activation.
Plate incorporation test:
Mutagenicity: In the absence of the metabolic activation system the test compound did not result in relevant increases in the number of revertants in any of the bacterial strains. Also in the presence of rat liver activation system (10 % (v/v)), treatment of the cells with Reaktiv Rot F66813 did not result in relevant increases in the number of revertant colonies.
Preincubation test:
In the absence and in the presence of hamster liver S9-mix (30 % (v/v)) using the preincubation method according to Prival Reaktiv Rot F66813 did not result in relevant increases in the number of revertant colonies with any of the tester strains.
Summarizing, it can be stated thatReaktiv Rot F66813isnot mutagenicin the standard plate test (Ames Test) and in the preincubation method according to Prival (8) at the dose levels investigated.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2003
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- according to Prival und Mitchell (azo dye)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- according to Prival and Mitchell (azo dye)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix (S9 fraction from uninduced hamster liver, sodium phosphate buffer, glucose-6-phosphate, glucose-6-phosphate-dehydrogenase, NADP, NADH, MgCl2, KCl and FMN)
- Test concentrations with justification for top dose:
- Concentration range in the dose range finding test (with and without metabolic activation): 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Concentration range in the main test (with and without metabolic activation): 100, 333, 1000, 3330 and 5000 µg/plate - Vehicle / solvent:
- The sample was dissolved in Milli-Q water, then filter (0.22 µm)-sterilized.
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 2-nitrofluorene
- sodium azide
- methylmethanesulfonate
- other: daunomycine; 2-aminoanthracene
- Details on test system and experimental conditions:
- Two independent mutagenicity tests were performed, a dose range finding test and a mutation assay.
METHOD OF APPLICATION: in agar (plate incorporation)
DURATION:
- Preincubation period: 30 min at 37° C by 70 rpm
- Exposure duration: 48 h at 37° C in the dark
After this period the revertant colonies (histidine independent for Salmonella typhimurium bacteria and triptophan independent for Escherichia coli) were counted automatically with a Protos 5000 colony counter or manually, if less than 40 colonies per plate were present.
DETERMINATION OF CYTOTOXICITY
The condition of the bacterial background lawn is evaluated both macroscopically and microscopically using a dissecting microscope. - Evaluation criteria:
- Cytotoxicity: The reduction of the bacterial background lawn, the increase in the size of the microclonies and the reduction of the revertant colonies were observed.
A test subsance is considered negative if the total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value and the negative response should be reproducible in at least one independently repeated experiment.
A test subsance is considered positive if it produces at least a three-fold (TA1535, TA1537, TA98 and WP2uvrA) or two-fold (TA100) dose-related increase in the number of revertants and the positive response should be reproducible in at least one independently repeated experiment.
- doubling of spontaneous mutation rate in at least one tester strains either with or without S9 - Statistics:
- NA
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with
- 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:
- Observations:
No increase in the number of revertant colonies was observed upon treatment with BRF 112-1 under all conditions tested.
BRF 112-1 did not precipitate in the top agar. Precipitation on the plates was not observed at the start or at the end of the incubation period in all tester strains.
The negative and strain-specific positive control values were within our laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for TA1537 in the absence of S9-mix (first experiment; positive control). However, since this value was just outside the limit of the range and a more than three-fold increase was observed compared to the solvent control, the validity of the test was considered to be not affected. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation
BRF 112-1 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. - Executive summary:
BRF 112-1 was tested in the Salmonella typhimurium reverse mutation assay with four histidine requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA. The test was performed in two independent experiments in the presence and absence of S9-mix.
In the dise range finding test, BRF 112-1 was tested up to concentrations of 5000 μg/plate in the absence and presence of S9 -mix in the strains TA100 and WP2uvrA. The test subsance did not precipitate on the plates at this dise level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.
In the first and in the second mutation assay, BRF 112-1 was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed.
BRF 112 -1 did not induce a dose-related, two-fold increase in the number of revertant colonies in strain TA100, or a dose-related, three-fold increase in the number of revertant colonies in strain TA1535, TA1537, TA98 or WR2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.
Based on the results of this study it is conducted that BRF 112 -1 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2007
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- human lymphocytes
- Species / strain / cell type:
- lymphocytes: human
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix (liver microsomes from Aroclor 1254 induced rats with a co-factor solution))
- Test concentrations with justification for top dose:
- Concentration range in the main test (without and with metabolic activation): 0.56 mg/ml Concentration range in the main test (without and with metabolic activation): 1.67 mg/ml Concentration range in the main test (without and with metabolic activation): 5.00 mg/ml
- Vehicle / solvent:
- cell culture medium (RPMI)
- Untreated negative controls:
- yes
- Remarks:
- culture medium
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- methylmethanesulfonate
- Details on test system and experimental conditions:
- All samples were transfered to a shaking water bath and incubated at 37 °C.
Exposure period (without and with metabolic activation): 3 hours - Evaluation criteria:
- - mitotic indices: were determined by counting and recording
- chromosome analyses: structural and numerical abberations with Nikon microscopes.
The following parameters were evaluated:
• the number of metaphases with numerical aberrations (< 46 chromosomes, > 46 chromosomes, tetraploidy, endoreduplication)
• the number of metaphases with structural aberrations, excluding gaps
• the number of chromatid-type aberrations, excluding gaps
• the number of chromosome-type aberrations, excluding gaps
• the number of gaps (chromatid- and isochromatid-type).
If there were more than one structural aberration of the same type within one metaphase, these aberrations were referred to as „multiple“
For statistical comparisons of the numbers of a specific aberration, such aberrations were counted as two aberrations.
A statistically significant increase in the number of metaphases with aberrations or a concentration-related increase in this number is considered as a positive result. However, a result can also be regarded as positive when other than merely statistical considerations, for example the kind of aberrations observed, are taken into account. - Statistics:
- The Chi2-Test (two-tailed, p=0.05) was used for the comparison between the negative control and the BRF 112-1 cultures. If the results were positive, comparisons were made separately between the negative control and each concentration. If conditions for the Chi2-Test were not met, Fisher’s Exact Test was used. Chi2-Test or Fisher’s Exact Test were also used for the comparison between the negative and the positive controls.
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- slight at highest tested concentration
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with
- 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:
- Observations:
Cytotoxicity
Slight cytotoxicity of the test substance was noted in the experiment without a metabolic activation system: The mitotic indices of the test substance treated cultures were between 71.4 % (high test substance concentration) and 95.7 % (medium test substance concentration) of those of the corresponding negative controls. No cytotoxicity occurred in the experiment with a metabolic activation system: The mitotic indices of the test substance treated cultures were between 139 % and 202 % of those of the corresponding negative controls.
Structural aberrations
No statistically significant increases in the number of metaphases with structural aberrations were noted in the experiment without the use of a metabolic activation system at any concentration analysed compared to the concurrent negative controls. All figures were within the range of historical negative controls.
In the experiment with the use of a metabolic activation system, there was a clear and statistically significant increase in the number of metaphases with structural aberrations at each concentration analysed compared to the concurrent negative controls. There was also a statistically significant increase in the number of chromatid-type aberrations at the lowest and the highest concentration tested (0.56 and 5.00 mg/mL medium). The increase in metaphases with structural aberrations was not concentration dependent. All figures were far outside the range of historical negative control data.
Gaps
No statistically significant increases in the number of gaps were noted in any experiment performed at any concentration analysed compared to the concurrent negative controls, regardless whether a metabolic activation system was used or not.
Numerical aberrations
No statistically significant differences in the number of metaphases with less than 46 chromosomes, more than 46 chromosomes, endopolyploid or polyploid cells were found compared to the concurrent negative controls in any experiment, regardless whether a metabolic activation system was used or not. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative without metabolic activation
positive with metabolic activation
The test substance induced structural chromosomal aberrations in cultured human lymphocytes when a metabolic activation system was used. Without metabolic activation it did not induce any chromosome mutations. - Executive summary:
The study was performed to determine possible mutagenic properties of BRF 112 -1 by means of an in vitro mammalian chromosome aberration test in human lymphocytes, according to the directive 2000/32/EC, B.10. and to the OECD Guideline 473.
A total of eight experiments was performed: four of them without and four with the use of a metabolic activation system (liver microsomes from Aroclor 1254 induced rats, with a co-factor solution). A concentration range between nominal 0.56 and 5.00 mg test substance per mL medium was tested.
Without the use of a metabolic activation system, the test substance caused slight cytotoxic effects at the highest concentration tested (5.00 mg/mL). No cytotoxicity was noted with the use of a metabolic activation system.
There was no evidence that the test substance caused numerical aberrations or gaps at any concentration tested, regardless whether a metabolic activation system was used or not.
No marked or statistically significant increase of the number of metaphases with structural aberrations was noted in the experiment without a metabolic activation system and an incubation period of 3 hours. However, a clear and statistically significant increase of the number of metaphases with structural aberrations was noted in the experiment with a metabolic activation system at each of the three test substance concentrations used. The figures were also far outside the range of historical negative control data.
In accordance with the test guidelines, no further experiments without or with the use of a metabolic activation system were therefore evaluated.
There was relevant evidence that test substance induced structural chromosomal aberrations in cultured human lymphocytes when a metabolic activation system was used. The conclusion is based on a clear and statistically significant increase of metaphases with structural aberrations in the experiment with a metabolic activation system and on the finding that all figures were far outside the range of historical negative controls.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see attachement section 13
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attachement section 13
3. ANALOGUE APPROACH JUSTIFICATION
see attachement section 13 - Reason / purpose for cross-reference:
- read-across source
- GLP compliance:
- yes (incl. QA statement)
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (pH 7.32 measured in the solvent control versus pH 7.36 measured at 8590 µg/mL)
- Effects of osmolality: no relevant increase (287 measured in the solvent control versus 329 measured at 8590 µg/mL)
- Evaporation from medium: Not examined
- Water solubility:123g (without correction for purity)
- Precipitation: No precipitation of the test item was observed up to the maximum concentration in all experiments.
- Other confounding effects: None
RANGE-FINDING/SCREENING STUDIES:
According to the current OECD Guideline for Cell Gene Mutation Tests at least four analysable concentrations should be used in two parallel cultures. For freely-soluble and non-cytotoxic test items the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest. For cytotoxic test items the maximum concentration should result in approximately 10 to 20% relative survival or cell density at subcultivation and the analysed concentrations should cover a range from the maximum to little or no cytotoxicity. Relatively insoluble test items should be tested up to the highest concentration that can be formulated in an appropriate solvent as solution or homogenous suspension. These test items should be tested up or beyond their limit of solubility. Precipitation should be evaluated at the beginning and at the end of treatment by the unaided eye.
The range finding pre-experiment was performed using a concentration range of 67.1 to 8590 µg/mL to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24-hours treatment) of metabolic activation. The highest applied concentration in the pre-test on toxicity (8590 µg/ml) was equal to 5 mg/mL of the pure substance.
Relevant toxic effects occurred after 4 hours treatment at 4295 µg/mL and above with metabolic activation. Following 4 hours treatment without metabolic activation toxic effects were noted at 2147.5 µg/mL and above. Another low value of the cloning efficiency was noted at 536.9 µg/mL following 4-hour treatment without metabolic activation. This effect was judged as irrelevant fluctuation rather than a true cytotoxic effect however, as the relative cloning efficiency remained above 50% at the next higher concentration. Following 24 hours treatment without metabolic activation a strong toxic effect occurred at 536.9 µg/mL. At all higher concentrations the cell growth was completely inhibited.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. No precipitation or phase separation was observed up to the maximum concentration with and without metabolic activation following 4 and 24 hours treamtment.
There was no relevant shift of the osmolarity and pH value of the medium even at the maximum concentration of the test item.
The dose range of the first experiment was set according to the data generated in the pre-experiment. The dose range of the second experiment was adjusted to data produced in the pre-experiment (without metabolic activation) and in the first experiment (with metabolic activation). The individual concentrations were generally spaced by a factor of 2.0. A narrower spacing was used at higher concentrations to cover the cytotoxic range more closely.
To overcome problems with possible deviations in toxicity the main experiments were started with more than four concentrations.
COMPARISON WITH HISTORICAL CONTROL DATA: Complies
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects, indicated by a relative cloning efficiency I or a relative cell density at first subcultivation of less than 50% in both parallel cultures, occurred in the first experiment at 1050 µg/mL and above without metabolic activation. In the second experiment relevant cytotoxic effects as described above were noted at 805.3 µg/mL and above without metabolic activation and at 4295 µg/mL with metabolic activation. The recommended cytotoxic range of approximately 10%-20% relative cloning efficiency or relative cell density was covered with and without metabolic activation. The difference in cytotoxicity noted in the first and the second experiment with metabolic activation is based on the variability of the cell density during treatment. According to the OECD 476 guideline proliferating cells should be treated so, the actual cell density varies from experiment to experiment. - Conclusions:
- In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore,the test substance is considered to be non-mutagenic in this HPRT assay. - Executive summary:
The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.
The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
The main experiments were evaluated at the following concentrations:
exposure
periodS9
mixconcentrations
in µg/mLExperiment I
4 hours
-
525.0
1050
2100
3150
4200
4 hours
+
1050
2100
4200
6300
8400
24 hours
-
134.2
268.4
536.9
805.3
1073.8
4 hours
+
268.4
536.9
1073.6
2147.5
4295.0
No precipitation of the test item was observed up to the maximum concentration in any of the experiments.
Relevant cytotoxic effects, indicated by a relative cloning efficiency I or a relative cell density at first subcultivation of less than 50% in both parallel cultures, occurred in the first experiment at 1050 µg/mL and above without metabolic activation. In the second experiment relevant cytotoxic effects as described above were noted at 805.3 µg/mL and above without metabolic activation and at 4295 µg/mL with metabolic activation. The recommended cytotoxic range of approximately 10%-20% relative cloning efficiency or relative cell density was covered with and without metabolic activation. The difference in cytotoxicity noted in the first and the second experiment with metabolic activation is based on the variability of the cell density during treatment. According to the OECD 476 guideline proliferating cells should be treated so, the actual cell density varies from experiment to experiment.
No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments up to the maximum concentration. The mutation frequency did not exceed the historical range of solvent controls, the induction factor did not reach or exceed the threshold of 3.0.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 5.5 up to 23.4 mutants per 106cells; the range of the groups treated with the test item was from 4.5 up to 35.4 mutants per 106cells.
EMS (150 µg/mL) and DMBA (1.1 µg/mL in experiment I and 2.2 µg/mL in experiment II) were used as positive controls and showed a distinct increase in induced mutant colonies.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2014-04-23 until 2014-07-08
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Principles of method if other than guideline:
- first experiment 4 hours treatment with and without metabolic activation
second experiment 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation - GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- HPRT
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
- Test concentrations with justification for top dose:
- Experiment I:
without metabolic activation: 525.0; 1050; 2100; 3150; 4200 µg/mL
with metabolic activation: 1050; 2100; 4200; 6300; 8400 µg/mL
Experiment II:
without metabolic activation: 134.2; 268.4; 536.9; 805.3; 1073.8 µg/mL
with metabolic activation: 268.4; 536.9; 1073.6; 2147.5; 4295.0 µg/mL - Vehicle / solvent:
- deionised water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days
SELECTION AGENT (mutation assays): 6-Thioguanine
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: >1,5x10exp. 6
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation fre¬quency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data. - Statistics:
- A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (pH 7.32 measured in the solvent control versus pH 7.36 measured at 8590 µg/mL)
- Effects of osmolality: no relevant increase (287 measured in the solvent control versus 329 measured at 8590 µg/mL)
- Evaporation from medium: Not examined
- Water solubility:123g (without correction for purity)
- Precipitation: No precipitation of the test item was observed up to the maximum concentration in all experiments.
- Other confounding effects: None
RANGE-FINDING/SCREENING STUDIES:
According to the current OECD Guideline for Cell Gene Mutation Tests at least four analysable concentrations should be used in two parallel cultures. For freely-soluble and non-cytotoxic test items the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest. For cytotoxic test items the maximum concentration should result in approximately 10 to 20% relative survival or cell density at subcultivation and the analysed concentrations should cover a range from the maximum to little or no cytotoxicity. Relatively insoluble test items should be tested up to the highest concentration that can be formulated in an appropriate solvent as solution or homogenous suspension. These test items should be tested up or beyond their limit of solubility. Precipitation should be evaluated at the beginning and at the end of treatment by the unaided eye.
The range finding pre-experiment was performed using a concentration range of 67.1 to 8590 µg/mL to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24-hours treatment) of metabolic activation. The highest applied concentration in the pre-test on toxicity (8590 µg/ml) was equal to 5 mg/mL of the pure substance.
Relevant toxic effects occurred after 4 hours treatment at 4295 µg/mL and above with metabolic activation. Following 4 hours treatment without metabolic activation toxic effects were noted at 2147.5 µg/mL and above. Another low value of the cloning efficiency was noted at 536.9 µg/mL following 4-hour treatment without metabolic activation. This effect was judged as irrelevant fluctuation rather than a true cytotoxic effect however, as the relative cloning efficiency remained above 50% at the next higher concentration. Following 24 hours treatment without metabolic activation a strong toxic effect occurred at 536.9 µg/mL. At all higher concentrations the cell growth was completely inhibited.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. No precipitation or phase separation was observed up to the maximum concentration with and without metabolic activation following 4 and 24 hours treamtment.
There was no relevant shift of the osmolarity and pH value of the medium even at the maximum concentration of the test item.
The dose range of the first experiment was set according to the data generated in the pre-experiment. The dose range of the second experiment was adjusted to data produced in the pre-experiment (without metabolic activation) and in the first experiment (with metabolic activation). The individual concentrations were generally spaced by a factor of 2.0. A narrower spacing was used at higher concentrations to cover the cytotoxic range more closely.
To overcome problems with possible deviations in toxicity the main experiments were started with more than four concentrations.
COMPARISON WITH HISTORICAL CONTROL DATA: Complies
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects, indicated by a relative cloning efficiency I or a relative cell density at first subcultivation of less than 50% in both parallel cultures, occurred in the first experiment at 1050 µg/mL and above without metabolic activation. In the second experiment relevant cytotoxic effects as described above were noted at 805.3 µg/mL and above without metabolic activation and at 4295 µg/mL with metabolic activation. The recommended cytotoxic range of approximately 10%-20% relative cloning efficiency or relative cell density was covered with and without metabolic activation. The difference in cytotoxicity noted in the first and the second experiment with metabolic activation is based on the variability of the cell density during treatment. According to the OECD 476 guideline proliferating cells should be treated so, the actual cell density varies from experiment to experiment. - Conclusions:
- In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, SA01 is considered to be non-mutagenic in this HPRT assay. - Executive summary:
The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.
The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
The main experiments were evaluated at the following concentrations:
exposure
periodS9
mixconcentrations
in µg/mLExperiment I
4 hours
-
525.0
1050
2100
3150
4200
4 hours
+
1050
2100
4200
6300
8400
24 hours
-
134.2
268.4
536.9
805.3
1073.8
4 hours
+
268.4
536.9
1073.6
2147.5
4295.0
No precipitation of the test item was observed up to the maximum concentration in any of the experiments.
Relevant cytotoxic effects, indicated by a relative cloning efficiency I or a relative cell density at first subcultivation of less than 50% in both parallel cultures, occurred in the first experiment at 1050 µg/mL and above without metabolic activation. In the second experiment relevant cytotoxic effects as described above were noted at 805.3 µg/mL and above without metabolic activation and at 4295 µg/mL with metabolic activation. The recommended cytotoxic range of approximately 10%-20% relative cloning efficiency or relative cell density was covered with and without metabolic activation. The difference in cytotoxicity noted in the first and the second experiment with metabolic activation is based on the variability of the cell density during treatment. According to the OECD 476 guideline proliferating cells should be treated so, the actual cell density varies from experiment to experiment.
No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments up to the maximum concentration. The mutation frequency did not exceed the historical range of solvent controls, the induction factor did not reach or exceed the threshold of 3.0.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 5.5 up to 23.4 mutants per 106cells; the range of the groups treated with the test item was from 4.5 up to 35.4 mutants per 106cells.
EMS (150 µg/mL) and DMBA (1.1 µg/mL in experiment I and 2.2 µg/mL in experiment II) were used as positive controls and showed a distinct increase in induced mutant colonies.
Referenceopen allclose all
Summary Table
| relative | relative | relative | mutant | relative | relative | relative | mutant | |||||
| conc. | S9 | cloning | cell | cloning | colonies/ | induction | cloning | cell | cloning | colonies/ | induction | |
| µg/mL | mix | efficiency I | density | efficiency II | 106cells | factor | efficiency I | density | efficiency II | 106cells | factor | |
| % | % | % | % | % | % | |||||||
| Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| Experiment I / 4 h treatment | culture I | culture II | ||||||||||
| Solvent control with water | - | 100.0 | 100.0 | 100.0 | 10.1 | 1.0 | 100.0 | 100.0 | 100.0 | 12.4 | 1.0 | |
| Positive control (EMS) | 150.0 | - | 90.6 | 77.6 | 99.9 | 188.3 | 18.7 | 97.8 | 55.3 | 87.1 | 221.4 | 17.8 |
| Test item | 131.3 | - | 55.9 | culture was not continued# | 79.5 | culture was not continued# | ||||||
| Test item | 262.5 | - | 63.4 | culture was not continued# | 68.7 | culture was not continued# | ||||||
| Test item | 525.0 | - | 49.9 | 93.0 | 111.9 | 8.3 | 0.8 | 67.6 | 69.6 | 105.6 | 4.9 | 0.4 |
| Test item | 1050.0 | - | 29.6 | 93.0 | 104.6 | 5.9 | 0.6 | 38.2 | 81.5 | 93.4 | 12.2 | 1.0 |
| Test item | 2100.0 | - | 32.2 | 95.4 | 90.8 | 21.6 | 2.1 | 31.2 | 91.8 | 100.1 | 19.9 | 1.6 |
| Test item | 3150.0 | - | 9.1 | 83.8 | 96.4 | 4.6 | 0.5 | 16.8 | 76.9 | 96.5 | 5.6 | 0.4 |
| Test item | 4200.0 | - | 8.1 | 76.5 | 94.8 | 17.6 | 1.7 | 10.0 | 66.3 | 85.7 | 21.6 | 1.7 |
| Solvent control with water | + | 100.0 | 100.0 | 100.0 | 16.3 | 1.0 | 100.0 | 100.0 | 100.0 | 7.3 | 1.0 | |
| Positive control (DMBA) | 1.1 | + | 99.7 | 99.9 | 93.8 | 131.5 | 8.1 | 89.2 | 100.6 | 95.2 | 144.6 | 19.7 |
| Test item | 262.5 | + | 82.5 | culture was not continued# | 100.4 | culture was not continued# | ||||||
| Test item | 525.0 | + | 92.9 | culture was not continued# | 69.5 | culture was not continued# | ||||||
| Test item | 1050.0 | + | 81.4 | 120.2 | 76.2 | 4.5 | 0.3 | 63.6 | 130.1 | 50.6 | 6.9 | 0.9 |
| Test item | 2100.0 | + | 89.2 | 109.1 | 76.6 | 18.1 | 1.1 | 78.3 | 107.1 | 93.6 | 19.2 | 2.6 |
| Test item | 4200.0 | + | 89.0 | 121.4 | 107.7 | 9.9 | 0.6 | 59.1 | 93.5 | 125.9 | 16.6 | 2.3 |
| Test item | 6300.0 | + | 69.2 | 107.4 | 120.2 | 16.1 | 1.0 | 60.1 | 97.4 | 129.3 | 13.3 | 1.8 |
| Test item | 8400.0 | + | 62.0 | 94.6 | 100.0 | 9.4 | 0.6 | 47.5 | 88.4 | 99.1 | 11.2 | 1.5 |
| Experiment II / 24 h treatment | culture I | culture II | ||||||||||
| Solvent control with water | - | 100.0 | 100.0 | 100.0 | 5.5 | 1.0 | 100.0 | 100.0 | 100.0 | 23.4 | 1.0 | |
| Positive control (EMS) | 150.0 | - | 95.1 | 84.4 | 98.3 | 450.7 | 82.4 | 96.1 | 83.9 | 78.3 | 639.7 | 27.3 |
| Test item | 33.5 | - | 90.6 | culture was not continued# | 95.7 | culture was not continued# | ||||||
| Test item | 67.1 | - | 93.1 | culture was not continued# | 94.9 | culture was not continued# | ||||||
| Test item | 134.2 | - | 93.1 | 63.8 | 100.8 | 13.6 | 2.5 | 92.5 | 69.5 | 91.5 | 21.9 | 0.9 |
| Test item | 268.4 | - | 95.8 | 67.7 | 108.6 | 8.4 | 1.5 | 84.2 | 50.9 | 89.5 | 9.9 | 0.4 |
| Test item | 536.9 | - | 88.4 | 56.9 | 96.8 | 7.9 | 1.5 | 83.2 | 59.8 | 87.3 | 35.4 | 1.5 |
| Test item | 805.3 | - | 10.3 | 51.9 | 101.3 | 10.0 | 1.8 | 8.8 | 40.7 | 88.4 | 31.4 | 1.3 |
| Test item | 1073.8 | - | 0.0 | 42.0 | 101.5 | 10.8 | 2.0 | 0.0 | 48.8 | 93.2 | 16.5 | 0.7 |
| Experiment II / 4 h treatment | ||||||||||||
| Solvent control with water | + | 100.0 | 100.0 | 100.0 | 17.8 | 1.0 | 100.0 | 100.0 | 100.0 | 20.1 | 1.0 | |
| Positive control (DMBA) | 2.2 | + | 98.9 | 99.6 | 105.1 | 184.1 | 10.3 | 100.3 | 58.2 | 93.6 | 264.7 | 13.2 |
| Test item | 268.4 | + | 87.9 | 86.9 | 96.3 | 19.4 | 1.1 | 105.6 | 104.8 | 99.0 | 20.5 | 1.0 |
| Test item | 536.9 | + | 91.2 | 124.3 | 93.6 | 15.4 | 0.9 | 99.9 | 107.7 | 100.5 | 15.5 | 0.8 |
| Test item | 1073.8 | + | 99.7 | 116.2 | 93.6 | 14.9 | 0.8 | 95.2 | 100.3 | 100.8 | 8.9 | 0.4 |
| Test item | 2147.5 | + | 96.6 | 93.0 | 95.9 | 11.6 | 0.7 | 84.2 | 97.1 | 97.4 | 23.1 | 1.1 |
| Test item | 4295.0 | + | 16.5 | 36.5 | 95.6 | 35.0 | 2.0 | 15.4 | 42.4 | 96.0 | 8.0 | 0.4 |
| Test item | 6442.5 | + | 0.0 | 3.8 | culture was not continued## | 0.0 | 4.5 | culture was not continued## | ||||
| Test item | 8590.0 | + | 0.0 | culture was not continued## | 0.0 | culture was not continued## | ||||||
# culture was not continued since a minimum of only four analysable concentrations is required
## culture was not continued due to exceedingly severe cytotoxic effects
Summary Table
| relative | relative | relative | mutant | relative | relative | relative | mutant | |||||
| conc. | S9 | cloning | cell | cloning | colonies/ | induction | cloning | cell | cloning | colonies/ | induction | |
| µg/mL | mix | efficiency I | density | efficiency II | 106cells | factor | efficiency I | density | efficiency II | 106cells | factor | |
| % | % | % | % | % | % | |||||||
| Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| Experiment I / 4 h treatment | culture I | culture II | ||||||||||
| Solvent control with water | - | 100.0 | 100.0 | 100.0 | 10.1 | 1.0 | 100.0 | 100.0 | 100.0 | 12.4 | 1.0 | |
| Positive control (EMS) | 150.0 | - | 90.6 | 77.6 | 99.9 | 188.3 | 18.7 | 97.8 | 55.3 | 87.1 | 221.4 | 17.8 |
| Test item | 131.3 | - | 55.9 | culture was not continued# | 79.5 | culture was not continued# | ||||||
| Test item | 262.5 | - | 63.4 | culture was not continued# | 68.7 | culture was not continued# | ||||||
| Test item | 525.0 | - | 49.9 | 93.0 | 111.9 | 8.3 | 0.8 | 67.6 | 69.6 | 105.6 | 4.9 | 0.4 |
| Test item | 1050.0 | - | 29.6 | 93.0 | 104.6 | 5.9 | 0.6 | 38.2 | 81.5 | 93.4 | 12.2 | 1.0 |
| Test item | 2100.0 | - | 32.2 | 95.4 | 90.8 | 21.6 | 2.1 | 31.2 | 91.8 | 100.1 | 19.9 | 1.6 |
| Test item | 3150.0 | - | 9.1 | 83.8 | 96.4 | 4.6 | 0.5 | 16.8 | 76.9 | 96.5 | 5.6 | 0.4 |
| Test item | 4200.0 | - | 8.1 | 76.5 | 94.8 | 17.6 | 1.7 | 10.0 | 66.3 | 85.7 | 21.6 | 1.7 |
| Solvent control with water | + | 100.0 | 100.0 | 100.0 | 16.3 | 1.0 | 100.0 | 100.0 | 100.0 | 7.3 | 1.0 | |
| Positive control (DMBA) | 1.1 | + | 99.7 | 99.9 | 93.8 | 131.5 | 8.1 | 89.2 | 100.6 | 95.2 | 144.6 | 19.7 |
| Test item | 262.5 | + | 82.5 | culture was not continued# | 100.4 | culture was not continued# | ||||||
| Test item | 525.0 | + | 92.9 | culture was not continued# | 69.5 | culture was not continued# | ||||||
| Test item | 1050.0 | + | 81.4 | 120.2 | 76.2 | 4.5 | 0.3 | 63.6 | 130.1 | 50.6 | 6.9 | 0.9 |
| Test item | 2100.0 | + | 89.2 | 109.1 | 76.6 | 18.1 | 1.1 | 78.3 | 107.1 | 93.6 | 19.2 | 2.6 |
| Test item | 4200.0 | + | 89.0 | 121.4 | 107.7 | 9.9 | 0.6 | 59.1 | 93.5 | 125.9 | 16.6 | 2.3 |
| Test item | 6300.0 | + | 69.2 | 107.4 | 120.2 | 16.1 | 1.0 | 60.1 | 97.4 | 129.3 | 13.3 | 1.8 |
| Test item | 8400.0 | + | 62.0 | 94.6 | 100.0 | 9.4 | 0.6 | 47.5 | 88.4 | 99.1 | 11.2 | 1.5 |
| Experiment II / 24 h treatment | culture I | culture II | ||||||||||
| Solvent control with water | - | 100.0 | 100.0 | 100.0 | 5.5 | 1.0 | 100.0 | 100.0 | 100.0 | 23.4 | 1.0 | |
| Positive control (EMS) | 150.0 | - | 95.1 | 84.4 | 98.3 | 450.7 | 82.4 | 96.1 | 83.9 | 78.3 | 639.7 | 27.3 |
| Test item | 33.5 | - | 90.6 | culture was not continued# | 95.7 | culture was not continued# | ||||||
| Test item | 67.1 | - | 93.1 | culture was not continued# | 94.9 | culture was not continued# | ||||||
| Test item | 134.2 | - | 93.1 | 63.8 | 100.8 | 13.6 | 2.5 | 92.5 | 69.5 | 91.5 | 21.9 | 0.9 |
| Test item | 268.4 | - | 95.8 | 67.7 | 108.6 | 8.4 | 1.5 | 84.2 | 50.9 | 89.5 | 9.9 | 0.4 |
| Test item | 536.9 | - | 88.4 | 56.9 | 96.8 | 7.9 | 1.5 | 83.2 | 59.8 | 87.3 | 35.4 | 1.5 |
| Test item | 805.3 | - | 10.3 | 51.9 | 101.3 | 10.0 | 1.8 | 8.8 | 40.7 | 88.4 | 31.4 | 1.3 |
| Test item | 1073.8 | - | 0.0 | 42.0 | 101.5 | 10.8 | 2.0 | 0.0 | 48.8 | 93.2 | 16.5 | 0.7 |
| Experiment II / 4 h treatment | ||||||||||||
| Solvent control with water | + | 100.0 | 100.0 | 100.0 | 17.8 | 1.0 | 100.0 | 100.0 | 100.0 | 20.1 | 1.0 | |
| Positive control (DMBA) | 2.2 | + | 98.9 | 99.6 | 105.1 | 184.1 | 10.3 | 100.3 | 58.2 | 93.6 | 264.7 | 13.2 |
| Test item | 268.4 | + | 87.9 | 86.9 | 96.3 | 19.4 | 1.1 | 105.6 | 104.8 | 99.0 | 20.5 | 1.0 |
| Test item | 536.9 | + | 91.2 | 124.3 | 93.6 | 15.4 | 0.9 | 99.9 | 107.7 | 100.5 | 15.5 | 0.8 |
| Test item | 1073.8 | + | 99.7 | 116.2 | 93.6 | 14.9 | 0.8 | 95.2 | 100.3 | 100.8 | 8.9 | 0.4 |
| Test item | 2147.5 | + | 96.6 | 93.0 | 95.9 | 11.6 | 0.7 | 84.2 | 97.1 | 97.4 | 23.1 | 1.1 |
| Test item | 4295.0 | + | 16.5 | 36.5 | 95.6 | 35.0 | 2.0 | 15.4 | 42.4 | 96.0 | 8.0 | 0.4 |
| Test item | 6442.5 | + | 0.0 | 3.8 | culture was not continued## | 0.0 | 4.5 | culture was not continued## | ||||
| Test item | 8590.0 | + | 0.0 | culture was not continued## | 0.0 | culture was not continued## | ||||||
# culture was not continued since a minimum of only four analysable concentrations is required
## culture was not continued due to exceedingly severe cytotoxic effects
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
The test substance did not show genotoxic properties in an mouse micronucleus test in-vivo
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Mode of Action Analysis / Human Relevance Framework
Kirkland et al (2005) demonstrated an extremely high false-positive rate for in-vitro clastogenicity tests, particularly in mammalian cell tests, when compared to rodent carcinogenicity study results (Poth, A (2008)). “Certain characteristics of the commonly used rodent cell lines (CHO, CHL, V79, L5178Y, etc.), such as their p53 status, their karyotypic instability and their DNA repair deficiencies, are recognized as possibly contributing to the high rate of irrelevant positives. Also the need for exogenous metabolism with the cell systems is expected to contribute to these irrelevant positive findings, as metabolites produced by S9 used as a metabolic source in cell culture may be quite different from those produced by normal human liver metabolism.” Accordingly, “the high false-positive rate of the established in-vitro mammalian cell tests means that an increased number of compounds are subjected to earlier and additional in-vivo genotoxicity testing.”
In addition, it is well known that vinyl-sulphone compounds result in false positive test results in in-vitro tests for clastogenicity (Dearfield KL et al. (1991); Warra TJ et al. (1990)). This is due to the fact that these chemical agents react via the Michael addition reaction. Chemical reactivity via Michael addition is essential for many of the uses for which these compounds are important. As in the currently assessed dye, vinyl sulphone moieties are used in fiber-reactive dyes (MacGregor et at. (1980)). These compounds are known to deplete glutathione in in‑vitro test systems, in which the concentration of phase II enzymes is very low. Glutathione plays a role in the detoxification of many compounds. Conjugation with glutathione via Michael addition and subsequent excretion is the most common bio-elimination route for these compounds. Since in-vitro systems have low levels of glutathione, the glutathione depletion leads to a positive result in the in-vitro test system, which is not the case in the in-vivo test system, where glutathione is present in adequate amount, as could be shown in plenty of tests with vinyl sulphone dyes (internal data DyStar). Hence, the in-vivo test produces more reliable data for this kind of substance.
Dearfield KL et al. (1991). Genotoxicity in mouse lymphoma cells .of chemicals capable of Michael addition. Mutagenesis 1991;6(6):519-525
Kirkland et al (2005). Evaluation of the ability of a battery of three in-vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity.Mutat Res. 2005 July 4;584(1–2):1–256
MacGregor et at. (1980). Mutagenicity tests of fabric-finishing agents in Salmonella typhimurium: fiber-reactive wool dyes and cotton flame retardants. Environ. Mutagenesis 1980;2:405-418
Poth, A (2008). Challenges in Testing for Genotoxycity. Genetic Toxicology and Cell Biology, RCC Cytotest Cell Research GmbH
Warra TJ et al. (1990). Methyl vinyl sulphone: A new class of Michael-type genotoxin. Mutat Res. 1990;245:191-199
Additional information
The test substance was negative in various Ames tests with Salmonella and E. coli strains including the Prival modification. In addition, the structural analogue 01 was negative in the mutagenicity test in mammalian cells (HPRT test). Reactive Red F-66813 did not show any clastogenic effects in an in-vitro chromosome aberration test without metabolic activation system. However, it showed a slight clastogenic effect in this test in the presence of the metabolic activation system. This is in line with the result of many in-vitro clastogenicity tests for vinyl-sulphone dyes and reflects a falso positive response.
It is well known that vinyl-sulphone compounds result in false positive test results in in-vitro tests for clastogenicity (Dearfield KL et al. (1991); Warra TJ et al. (1990)). This is due to the fact that these chemical agents react via the Michael addition reaction. Chemical reactivity via Michael addition is essential for many of the uses for which these compounds are important. As in the currently assessed dye, vinyl sulphone moieties are used in fiber-reactive dyes (MacGregor et at. (1980)). These compounds are known to deplete glutathione in in‑vitro test systems, in which the concentration of phase II enzymes is very low. Glutathione plays a role in the detoxification of many compounds. Conjugation with glutathione via Michael addition and subsequent excretion is the most common bio-elimination route for these compounds. Since in-vitro systems have low levels of glutathione, the glutathione depletion leads to a positive result in the in-vitro test system, which is not the case in the in-vivo test system, where glutathione is present in adequate amount, as could be shown in the negative in-vivo micronucleus study.
Short description of key information:
The test item did not show genotoxic effects in in-vitro and in-vivo
genetic toxicity studies
Endpoint Conclusion: No adverse effect observed (negative)
Justification for classification or non-classification
The above studies have all been ranked reliability 1 according to the Klimisch et al system. This ranking was deemed appropriate because the studies were conducted to GLP an in compliance with agreed protocols. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds. As the effects are considered adaptive rather than toxicological, no classification is proposed.
The above results triggered no classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008). No classification for prolonged effects is therefore required.
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