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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The conclusion is based on three key studies that together address all potential mode-of-actions. All three studies are reliable and valid.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-12-05 to 2013-05-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study comparable to OECD TG 487 (2010)
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD TG 487 (2010)
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
A series of in-house non-GLP validation experiments was performed to get proper responses of statistical significance when using the specified positive controls. To achieve such response the test design, specifically for the treatment, the expression phase and harvest time, was slightly modified compared to the OECD Guideline 487. The optimum in responses was found with the following schedule, which was decided to be used in the main study:Experiment I: 4 hours pulse treatment (with and without metabolic activation), expression phase 16 hours, cytokinesis block 20 hours.Experiment II: 4 hours pulse treatment (with metabolic activation), expression phase 16 hours, cytokinesis block 20 hours. 20 hours continuous treatment (without metabolic activation), cytokinesis block 20 hours.
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
The occurrence of micronuclei in interphase blood lymphocytes provides an indirect but easy and rapid measure of chromosomal damage and aneugenicity.
Species / strain / cell type:
lymphocytes: blood samples
Details on mammalian cell type (if applicable):
- Type and identity of media: Blood samples were obtained from healthy, non-smoking donors not receiving medication. For this study, blood was collected from a female donor (24 years old) for Experiment I and from a female donor (34 years old) for Experiment II. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes. Blood samples were drawn by venous puncture and collected in heparinized tubes by Dr. V. Theodor (64380 Rossdorf, Germany). The tubes were sent to Harlan CCR to initiate cell cultures within 24 hrs after blood collection. The blood was stored before use at 4 °C.- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 obtained from phenobarbital/beta-naphthoflavone induced rats were used as the metabolic activation system.
Test concentrations with justification for top dose:
Experiment I: 32.5, 56.8, 99.5, 174.1, 304.6, 533.1, 932.9, 1632.7, 2857.1, 5000.0 µg/mLExperiment II: 1632.7, 2857.1, 5000.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water- Justification for choice of solvent/vehicle: test item soluble in water
Details on test system and experimental conditions:
METHOD OF APPLICATION: About 48 hours after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test group.DURATION- Preincubation period: approx. 48 hours- Exposure duration: 4 hours or 20 hours- Expression time (cells in growth medium)/Fixation time (start of exposure up to fixation or harvest of cells):4 hours of exposure + 16 hours of recovery + 20 hours with Cytochalasin B (4 µg/mL)20 hours of exposure + 20 hours with Cytochalasin B (4 µg/mL)SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B (4 µg/mL)STAIN (for cytogenetic assays): Giemsa (MERCK, 64293 Darmstadt, Germany)NUMBER OF REPLICATIONS: 2 per treatment and per sampling dateNUMBER OF CELLS EVALUATED: 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.DETERMINATION OF CYTOTOXICITY- Method: Cytokinesis-block proliferation index
Evaluation criteria:
A test item can be classified as non-mutagenic if:1. the number of micronucleated cells in all evaluated dose groups is in the range of the laboratory historical control data and/or2. no statistically significant or concentration-related increase in the number of micronucleated cells is observed.A test item can be classified as mutagenic if:1. the number of micronucleated cells is not in the range of the historical laboratory control data and2. either a concentration-related increase of micronucleated cells in three test groups or a statistically significant increase of the number of micronucleated cells is observed.
Statistics:
Statistical significance was confirmed by means of the Chi square test. However, both biological and statistical significance should be considered together. If the criteria for the test item mentioned above are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: noExp. I:Solvent control 7.65000 mg/L 7.6Exp. II:Solvent control 7.65000 mg/L 7.6- Effects of osmolality: noExp. I:Solvent control 282 mOsm5000 mg/L 355 mOsm/L2857.1 mg/L 329 mg/L1632.7 mOsm/LExp. II:Solvent control 283 mOsm5000 mg/L 355 mOsm/L2857.1 mg/L 326 mg/L- Evaporation from medium: not relevant- Water solubility: soluble up to and including 5 mg/mL- Precipitation: not observed- Other confounding effects:noRANGE-FINDING/SCREENING STUDIES:A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of reduction in the CBPI in comparison with the controls (% cytostasis) by counting 500 cells per culture in duplicate. The experimental conditions in this pre-experimental phase were identical to those required and described for the mutagenicity assay. The pre-experiment was performed with 10 concentrations of the test item and the solvent and positive controls. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix) and the cells were prepared 40 hrs after start of the exposure.COMPARISON WITH HISTORICAL CONTROL DATA: yes
Conclusions:
In a valid, reliable and conclusive study according to OECD TG 487 (2010), the test item Reaction mass of CXN1-55 did not induce micronuclei in human lymphocytes in vitro when tested up to the highest required concentration under the experimental conditions. Therefore, Reaction mass of CXN1-55 is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.
Executive summary:

In a valid, reliable and conclusive study according to OECD TG 487 (2010), the test item Reaction mass of CXN1-55, dissolved in deionised water, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments. The following study design was used:

Without S9-Mix With S9-Mix
Exp. I Exp. II Exp. I and II
Exposure period  4 hrs 20 hrs  4 hrs
Recovery 16 hrs - 16 hrs
Cytochalasin B exposure 20 hrs 20 hrs 20 hrs
Preparation interval 40 hrs 40 hrs 40 hrs
Total culture period 88 hrs 88 hrs 88 hrs

In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were evaluated for cytogenetic damage. No precipitation of the test item in the culture medium was observed.

No relevant cytotoxicity, indicated by reduced CBPI and described as cytostasis, could be observed up to the highest applied concentration either with or without metabolic activation.

The highest applied concentration in Experiment I (5000.0 µg/mL of the test item) was chosen with respect to the current OECD Guideline 487.

In the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. In Experiment I with S9 mix two statistically significant increases (0.90 and 1.20 % micronucleated cells) were observed after treatment with 2857.1 and 5000.0 µg/mL. Since these values are clearly in the range of the historical solvent control data (0.20 – 1.70 % micronucleated cells) the findings are not biologically relevant.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

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:
2012-11-02 to 2012-12-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study under GLP according to OECD TG 471 (1997) and EU Method B13/14 (2008)
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella typhimurium Strains:The histidine dependent strains are derived from Salmonella typhimurium strain LT2 through a mutation in the histidine locus. 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 and TA 100 the R-factor plasmid pKM 101 carries the ampicillin resistance marker.Escherichia coli Strain:Strain WP2 and its derivatives all carry the same defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent (Trp+) mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagen which substitute one base for another. Additionally, the uvrA derivative is deficient in the DNA repair process (excision repair damage). Such a repair-deficient strain may be more readily mutated by agents.The bacterial strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA were obtained from Trinova Biochem GmbH (35394 Gießen, Germany).Salmonella typhimuriumStrainsGenotypeType of mutations indicatedTA 1537his C 3076; rfa-; uvrB- frame shift mutationsTA 98his D 3052; rfa-; uvrB-; R-factorframe shift mutationsTA 1535his G 46; rfa-; uvrB- base-pair substitutionsTA 100his G 46; rfa-; uvrB-; R-factorbase-pair substitutionsEscherichia coliWP2uvrA trp-; uvrA- base-pair substitutions and others
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
- Type and identity of media:Storage: The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, 64293 Darmstadt/Germany) in liquid nitrogen.Precultures: From the thawed ampoules of the strains 0.5 mL suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. A solution of 20 ML ampicillin (25 Mg/mL) was added to the strains TA 98 and TA 100. This nutrient medium contains per litre:8 g Nutrient Broth (MERCK, 64293 Darmstadt/Germany)5 g NaCl (MERCK, 64293 Darmstadt/Germany)The bacterial cultures were incubated in a shaking water bath for 4 hours at 37° C. The optical density of the bacteria was determined by absorption measurement and the obtained values indicated that the bacteria were harvested at the late exponential or early stationary phase (108-109 cells/mL).Selective Agar: The plates with the selective agar were obtained from E. Merck, 64293 Darmstadt/Germany.Overlay Agar: The overlay agar contains per litre:for Salmonella typhimurium:7.0 g Agar Agar (MERCK, 64293 Darmstadt/Germany)6.0 g NaCl (MERCK, 64293 Darmstadt/Germany)10.5 mg L-Histidine×HCl×H2O (MERCK, 64293 Darmstadt/Germany)12.2 mg Biotin (MERCK, 64293 Darmstadt/Germany)for Escherichia coli:7.0 g Agar Agar (MERCK, 64293 Darmstadt/Germany)6.0 g NaCl (MERCK, 64293 Darmstadt/Germany)10.2 mg Tryptophan (MERCK, 64293 Darmstadt/Germany)Sterilisations were performed at 121 °C in an autoclave.- Properly maintained: yes (Regular checking of the properties of the strains regarding the membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in Harlan CCR according to B. Ames et al. and D. Maron and B. Ames. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.- Periodically checked for Mycoplasma contamination: not reported- Periodically checked for karyotype stability: not reported- Periodically "cleansed" against high spontaneous background: not reported
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
beta-naphtoflavone/phenobarbital induced rat liver S9 (Preparation by Harlan CCR)
Test concentrations with justification for top dose:
experiment 1 (pre-experiment): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plateexperiment 2: 33, 100, 333, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: none
Untreated negative controls:
yes
Remarks:
deionosed water
Negative solvent / vehicle controls:
no
Remarks:
deionosed water
True negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubationDURATION- Preincubation period:In the pre-incubation assay 100 ML test solution (solvent or reference mutagen solution (positive control)), 500 ML S9 mix / S9 mix substitution buffer and 100 ML bacterial suspension were mixed in a test tube and shaken at 37 °C for 60 minutes.- Exposure duration:After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.- Expression time (cells in growth medium):The optical density of the bacteria was determined by absorption measurement and the obtained values indicated that the bacteria were harvested at the late exponential or early stationary phase (108-109 cells/mL).- Selection time (if incubation with a selection agent): no selection agent used- Fixation time (start of exposure up to fixation or harvest of cells): no fixation usedNUMBER OF REPLICATIONS: 3 per treatmentDETERMINATION OF CYTOTOXICITY- Method: the toxicity of the test item results in a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn.OTHER EXAMINATIONS:- Other: The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB9 7BN, UK) with the software program Ames Study Manager. The counter was connected to a PC with printer to print out the individual values and the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS. noRANGE-FINDING/SCREENING STUDIES: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plateCOMPARISON WITH HISTORICAL CONTROL DATA: see belowADDITIONAL INFORMATION ON CYTOTOXICITY:
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1 Results Pre-Experiment and Experiment I

Metabolic activation test group Dose level (per plate) unit Revertant Colony Counts (Mean ±SD)
TA 1535 TA 1537 TA 98 TA 100
mean ± SD mean ± SD mean ± SD mean ± SD
Without deionised water     17 ± 2 17 ± 3 24 ± 4 140 ± 10
Untreated     17 ± 5 13 ± 3 24 ± 4 134 ± 2
Test item 3 µg 19 ± 6 18 ± 1 25 ± 5 127 ± 14
Test item 10 µg 16 ± 4 15 ± 2 22 ± 5 129 ± 10
Test item 33 µg 15 ± 4 17 ± 0 20 ± 4 130 ± 16
Test item 100 µg 16 ± 3 16 ± 4 19 ± 3 146 ± 23
Test item 333 µg 11 ± 2 15 ± 3 26 ± 7 126 ± 6
Test item 1000 µg 16 ± 4 18 ± 6 23 ± 4 140 ± 5
Test item 2500 µg 11 ± 4 12 ± 6 23 ± 3 133 ± 8
Test item 5000 µg 15 ± 7 11 ± 3 22 ± 7 136 ± 8
NaN3 10 µg 2261 ± 48         1933 ± 36
4-NOPD 10 µg         290 ± 9  
4-NOPD 50 µg     86 ± 4      
MMS 3 µL                        
Metabolic activation test group Dose level (per plate) unit Revertant Colony Counts (Mean ±SD)
TA 1535 TA 1537 TA 98 TA 100
mean ± SD mean ± SD mean ± SD mean ± SD
With deionised water     20 ± 2 19 ± 2 35 ± 4 147 ± 8
Untreated     13 ± 1 17 ± 2 36 ± 9 164 ± 8
Test item 3 µg 19 ± 9 23 ± 3 41 ± 4 157 ± 14
Test item 10 µg 18 ± 3 19 ± 6 37 ± 4 158 ± 4
Test item 33 µg 18 ± 4 16 ± 6 35 ± 12 158 ± 10
Test item 100 µg 20 ± 9 21 ± 1 30 ± 6 164 ± 18
Test item 333 µg 17 ± 2 17 ± 2 35 ± 2 165 ± 7
Test item 1000 µg 20 ± 3 19 ± 5 36 ± 5 160 ± 15
Test item 2500 µg 17 ± 5 19 ± 5 35 ± 9 170 ± 12
Test item 5000 µg 18 ± 5 24 ± 4 34 ± 4 162 ± 12
2-AA 2.5 µg 398 ± 8 240 ± 25 1615 ± 61 2142 ± 116
2-AA 10 µg                        

Table 2 Results Experiment II

Metabolic activation test group Dose level (per plate) unit Revertant Colony Counts (Mean ±SD)
TA 1535 TA 1537 TA 98 TA 100
mean ± SD mean ± SD mean ± SD mean ± SD
Without deionised water   17 ± 3 9 ± 3 25 ± 7 131 ± 25
Untreated   15 ± 5 11 ± 3 26 ± 3 131 ± 25
Test item 33 µg 16 ± 4 9 ± 4 27 ± 3 129 ± 3
Test item 100 µg 18 ± 2 8 ± 1 29 ± 3 121 ± 12
Test item 333 µg 18 ± 1 10 ± 3 27 ± 4 121 ± 6
Test item 1000 µg 13 ± 1 9 ± 4 33 ± 2 124 ± 22
Test item 2500 µg 15 ± 6 10 ± 3 30 ± 3 117 ± 14
Test item 5000 µg 16 ± 3 10 ± 2 29 ± 2 122 ± 6
NaN3 10 µg 1923 ± 103         1912 ± 67
4-NOPD 10 µg         317 ± 20    
4-NOPD 50 µg     66 ± 5        
MMS 3 µL                        
Metabolic activation test group Dose level (per plate) unit Revertant Colony Counts (Mean ±SD)
TA 1535 TA 1537 TA 98 TA 100
mean ± SD mean ± SD mean ± SD mean ± SD
With deionised water   23 ± 3 13 ± 4 35 ± 1 163 ± 2
Untreated   24 ± 3 13 ± 5 40 ± 6 167 ± 21
Test item 33 µg 22 ± 8 14 ± 3 27 ± 4 167 ± 5
Test item 100 µg 20 ± 3 14 ± 4 27 ± 6 158 ± 14
Test item 333 µg 20 ± 4 16 ± 3 30 ± 6 163 ± 3
Test item 1000 µg 17 ± 6 16 ± 1 34 ± 4 166 ± 2
Test item 2500 µg 19 ± 3 16 ± 2 31 ± 7 159 ± 17
Test item 5000 µg 20 ± 4 15 ± 3 32 ± 1 163 ± 10
2-AA 2.5 µg 243 ± 10 118 ± 11 1074 ± 51 1071 ± 98
2-AA 10 µg                        
Conclusions:
Interpretation of results (migrated information):negative with metabolic activationnegative without metabolic activationDuring the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pairchanges or frameshifts in the genome of the strains used.
Executive summary:

This valid, reliabale and conclusive study was performed to investigate the potential of Reaction mass of CXN1-55 to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. Testing was performed in

compliance to OECD guideline 471 and under GLP.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate

Experiment II: 33, 100, 333, 1000, 2500, and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tested strains was observed following treatment with Reaction mass of CXN1-55 at any dose level, neither in the presence nor 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 biological relevance.

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 item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, Reaction mass of CXN1-55 is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

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:
2013-03-22 to 2013-04-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study under GLP according to OECD TG 476 (1997)
Reason / purpose for cross-reference:
reference to same 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
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
gene for the functional HPRT enzyme: HPRT (hypoxanthine-guanine phosphoribosyl transferase) catalyzes the conversion of the nontoxic 6-TG (6-thioguanine) to its toxic ribophosphorylated derivative. Therefore, cells deficient in HPRT due to a forward mutation are resistant to 6-TG. These cells are able toproliferate in the presence of 6-TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 - 9 days. The expression period is terminated by adding 6-TG to the culture medium.
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of Harlan CCR allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants was depressed by treatment with HATmedium.- Properly maintained: yes- Periodically checked for Mycoplasma contamination: yes- Periodically checked for karyotype stability: yes- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 Mix: Phenobarbital/-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: none
Untreated negative controls:
yes
Remarks:
destilled water
Negative solvent / vehicle controls:
yes
Remarks:
destilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in mediumDURATION- Preincubation period: Two to three days after sub-cultivation stock cultures were trypsinized at 37 °C for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10 % FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2 % in PBS. The PBS is composed as follows (per litre):NaCl 8000 mgKCl 200 mgKH2PO4 200 mgNa2HPO4 150 mgPrior to the trypsin treatment the cells were rinsed with PBS buffer containing 200 mg/l EDTA (ethylene diamine tetraacetic acid). Approximately 1.5×106 (single culture) and 5×102 cells (in duplicate) were seeded in plastic culture flasks. The cells were grown for 24 hours prior to treatment. After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 Pl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel.- Exposure duration:Exposure 1: After 4 hours the medium was replaced with complete medium following two washing steps with "saline G".Exposure 2: In the second experiment the cells were exposed to the test item for 24 hours in complete medium, supplemented with 10 % FBS, in the absence of metabolic activation.- Expression time (cells in growth medium): Three or four days after treatment 1.5×106 cells per experimental point were subcultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5×105 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability.- Selection time (if incubation with a selection agent): For the selection of mutant cells the complete medium was supplemented with 11 Pg/mL 6-thioguanine. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution. The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.- Fixation time (start of exposure up to fixation or harvest of cells): The colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment.SELECTION AGENT (mutation assays): 6-thioguanineSPINDLE INHIBITOR (cytogenetic assays): not applicableSTAIN (for cytogenetic assays): 10 % methylene blue in 0.01 % KOH solutionNUMBER OF REPLICATIONS: 2NUMBER OF CELLS EVALUATED: approx. 500DETERMINATION OF CYTOTOXICITY- Method: cloning efficiency I (survival), cloning efficienty II (viablility), cell density, mutation rate
Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points. 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 non-mutagenic in this system.A positive response is described as follows: A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations 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. However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
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 were 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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

No relevant toxic effects occurred up to the maximum concentration of 5000 µg/mL.

No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The mutant frequency did not exceed the historical range of solvent controls.

The induction factor reached the threshold of three times the corresponding solvent control in the second culture of experiment II at 2500 µg/mL without metabolic activation. However, this increase was judged as biologically irrelevant as the absolute value of the mutation frequency (34.5 mutant colonies/106 cells) remained well within the historical range of solvent controls (2.6 - 40.3 mutant colonies/106 cells).

In the second culture of the second experiment with metabolic activation the threshold was exceeded at 312.5, 625, and 1250 µg/mL. These increases were based on the rather low solvent control of just 5.1 mutant colonies/106 cells and the historical range of solvent control (3.4 - 36.6 mutant colonies/106 cells) was not exceeded. Furthermore, there was no dose dependent trend as indicated by the lacking statistical significance.

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 5.1 up to 19.4 mutants per 106 cells; the range of the groups treated with the test item was from 2.7 up to 34.5 mutants per 106 cells.

EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusions:
In a valid, reliable and conclusive study according to OECD 476 (1997) and EU Method B.17 (2008), the test item did not induce gene mutations at the HPRT locus in V79 cells under the experimental conditions reported Therefore, Reaction mass of CXN1-55 is considered to be non-mutagenic in this HPRT assay.
Executive summary:

In a valid, reliable and conclusive study according to OECD 476 (1997) and EU Method B.17 (2008) and under GLP, the potential of Reaction mass of CXN1-55 to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster was investigated.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without metabolic activation (phenobarbital/beta-naphthoflavone induced rat liver S9 mix) and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The maximum concentration of the test item was 5 mg/mL in both experiments with and without metabolic activation. The tested concentrations ranged from 312.5 to 5000 µg/mL in all experiments. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The potential of Reaction mass of CXN1-55 to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA was investigated in a valid, reliable and conclusive study in compliance to OECD guideline 471 and under GLP: Neither genotoxic nor mutagenic with and without metabolic activation.

The potential of Reaction mass of CXN1-55 to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster was investigated In a valid, reliable and conclusive study according to OECD 476 (1997) and EU Method B.17 (2008) and under GLP: Neither genotoxic nor mutagenic with and without metabolic activation.

The potential of the test item Reaction mass of CXN1-55, dissolved in deionised water, to induce micronuclei in human lymphocytes in vitro in two independent experiments was investigated in a valid, reliable and conclusive study according to OECD TG 487 (2010) and under GLP: Neither genotoxic nor mutagenic with and without metabolic activation.

Justification for classification or non-classification

Based on the current data, CNX1 -55 is not classified for genotoxicity according to Regulation (EC) No. 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.