Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Taken together, it is concluded that the mutagenic response observed in the mouse lymphoma L5178Y test system is negative. Increases in the mutation frequency at the TK locus are only observed in the first experiment, and occur at severely cytotoxic concentrations that exceed the OECD guidance stating that the RTG should not go below 10%, since survival rates below 10% give rise to false positives. The mutation frequencies above the GEF + MF(controls) in the absence and presence of S9-mix were not confirmed in the second and third mutation experiment.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 June 2012 - 04 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Also complies with OECD GLP regulations. Deviations from guidelines were not deemed signicant.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
Also according to the recommendations of the "International Workshop on Genotoxicity Tests Workgroup" (IWGT) published in the literature (Clive et al., 1995, Moore et al. 1999, 2000, 2002, 2003, 2006, and 2007).
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): S-10793
- IUPAC nomenclature - Sodium diisobutyldithiophosphinate
- Lot S-20227-170B
- Appearance - White powder with lumps
- CAS No. 13360-78-6
- Molecular Formula - C8H18PS2.Na
- Molecular Weight - 232 g/mole
- Purity 93-94%
- Expiration date of the lot/batch: 16 December 2013
- Storage condition of test material: At room temperature in the dark
Target gene:
Thymidine Kinase (TK): the objective of the study was to evaluate the induction of forward mutations at the TK Locus (TK =/- to TK -/-)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) (Invitrogen Corporation) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively) (Invitrogen), 1 mM sodium pyruvate (Sigma) and 2 mM L-glutamin (Invitrogen Corporation).

Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium). (Horse serum was inactivated by incubation at 56 °C for at least 30 minutes.)

Exposure medium: For 3 hour exposure: Cells were exposed to the test substance in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium). For 24 hour exposure: Cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).

Selective medium: Basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 µg/ml trifluorothymidine (TFT) (Sigma).

Non-selective medium: Basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20).

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: Not reported
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Mutational experiments:
S9 homogenate- Trinova Biochem GmbH, Giessen, Germany, prepared from Sprague Dawley rats dosed orally with phenobarbital (80mg/kg-bw) and Beta-naphthaflavone (100 mg/kg-bw).

Dose range finding test: S9 prepared in-house
Type and composition of metabolic activation system:
- source of S9: laboratory in-house
- method of preparation of S9 mix: The rats were orally dosed for three consecutive days with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight) in corn oil (they were denied access to food for 3 to 4 hours preceding each dosing). One day after the final exposure (24 h), the rats were sedated using oxygen/carbon dioxide and then killed by decapitation. The rats received a limited quantity of food during the night before sacrifice. The livers of the rats were removed aseptically, and washed in cold (0°C) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2-EDTA. Subsequently the livers were minced in a blender and homogenized in 3 volumes of phosphate buffer with a Potter homogenizer. The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampoules, which were stored in liquid nitrogen (-196°C) for a maximum of 1 year.

Both
- concentration or volume of S9 mix and S9 in the final culture medium 50% S-9 fraction in S-9 mix; ca. 16% S-9 mix (8% S9-fraction) in the exposure medium
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix, 3 hours treatment: 512, 170, 510, 765 and 1182 µg/mL
Without S9-mix, 24 hours treatment: 512, 170, 510, 765 and 1182 µg/ml

Additional dose range finding test:
Without S9-mix, 24 hours treatment: 0.1, 1, 10 and 50 µg/ml

Experiment 1:
Without S9-mix: 1, 3, 10, 33, 100, 560 and 850 μg/ml
With S9-mix: 3, 10, 33, 100, 333 and 1000 μg/ml

Experiment 2
Without S9-mix: : 0.1, 0.3, 0.6, 1, 3, 5, 9 and 12 µg/ml
With S9-mix: 372, 465, 558, 651, 837, 1023, 1116 and 1395 μg/ml

Experiment 3:

Without S9-mix: 10, 33, 100, 300, 400, 600, 650 and 700 μg/ml
With S9-mix: 333, 600, 700, 800, 1100, 1200, 1300 and 1400 μg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: At concentrations of 23.5 mg/ml and higher S-10793 was suspended in ethanol. At concentrations of 12 mg/ml and lower the test substance was dissolved in ethanol. Except in the second dose range finding test, where S-10793 was still a suspension at 11.75 mg/ml. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. S-10793 concentrations were used within 1.5 hours after preparation
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: Not reported
- Exposure duration: 3 or 24 hours: Cell cultures were exposed to S-10793 in exposure medium for 3 hours in the presence of S9-mix and for 3 and 24 hours in the absence of S9-mix. To obtain more information about the cytotoxicity of S-10793, an additional experiment was performed for the 24 hours treatment period in the absence of S9-mix. After exposure, the cells were separated from treatment solutions by 2 centrifugation steps (216 g, 8 min) each followed by removal of the supernatant.
- Expression time (cells in growth medium): For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 106 cells (if possible) were subcultured every day in order to maintain log phase growth.
- Selection time (if incubation with a selection agent): The microtiter plates for CEday2 and MF were incubated for 11 or 12 days.
- Fixation time (start of exposure up to fixation or harvest of cells): Not reported

SELECTION AGENT (mutation assays): 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well.
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma)

NUMBER OF REPLICATIONS: Not reported

NUMBER OF CELLS EVALUATED: For determination of the MF a total number of 9.6 x 105 cells/concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium; for the positive control groups (MMS and CP) a total number of 9.6 x 105 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). After staining, the plates for the CE day2 and MF were scored with the naked eye or with the microscope.

DETERMINATION OF CYTOTOXICITY
- Method: Mutation Frequency, Cloning Efficiency, Relative Survival, Relative Total Growth, Suspension of Growth, Relative Suspension of Growth, Growth Rate
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120%. An acceptable number of surviving cells (10^6) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 per 10^6 survivors, and for CP not below 700 per 10^6 survivors.

DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.
Statistics:
The cloning efficiency was determined by dividing the number of empty wells by the total number of wells. This value obtained is the P(0), the zero term of the Poisson distribution: P(0) = number of empty wells/total number of wells

The cloning efficiency (CE) is therefore: CE = -ln P(0)/number of cells plated per well

The relative survival (RS) in each treatment group was determined by comparing cloning efficiencies in treatment and control cultures: RS = [CE(test)/CE(controls)] x 100

The Relative Total Growth (RTG) was also calculated as the product of the cumulative relative suspension growth (RSG) and the relative cloning efficiency for each culture: RTG = RSG x RSday2/100

Suspension Growth (SG) = [Day 0 cell count/ (1.25 x 105) *] x [Day 1 cell count/(1.25 x 105) *] x [Day 2 cell count] * Or appropriate cell concentration

Relative Suspension Growth (RSG) = SG (test) / SG (controls) x 100

RSday2 = CE day2 (test) / CE day2 (controls) x 100

The growth rate, as an indicator of optimally growing cultures, was calculated for the solvent control cultures: Growth Rate (GR) for the 3 hours treatment= [Day 1 cell count/(1.25 x 105) *] x [Day 2 cell count/(1.25 x 105) *]

Growth Rate (GR) for the 24 hours treatment= [Day 0 cell count/(1.25 x 105) ] x [Day 1 cell count/(1.25 x 105) *] x [Day 2 cell count/(1.25 x 105) *] * Or appropriate cell concentration

The mutation frequency was expressed as the number of mutants per 106 viable cells. The plating efficiencies of both mutant and viable cells (CE day2) in the same culture were determined and the mutation frequency (MF) was calculated as follows: MF = {-ln P(0)/number of cells plated per well}/ CE day2 x 106

Small and large colony mutation frequencies were calculated in an identical manner.
Species / strain:
mouse lymphoma L5178Y cells
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: No
Solvent control: 7.2
2320 µg/ml: 7.3
- Effects of osmolality: No
Solvent control: 0.283 mOsm/kg
2320 µg/ml: 0.314 mOsm/kg
- Precipitation: No precipitation was observed up to and including the top dose of 1182 µg/mL

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 170 µg/mL and above in the absence of S9, 3 hours treatment; at the dose level of 1182 µg/mL in the presence of S9, 3 hours treatment; at dose levels of 1 µg/mL and above in the absence of S9, 24 hours treatment.


COMPARISON WITH HISTORICAL CONTROL DATA:
- The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The meaningful increases in the mutation frequency at the TK locus were only observed at severely cytotoxic concentrations and mutation frequencies above the GEF + MF(controls) in the absence and presence of S9-mix were not confirmed in the second mutation experiment.

ADDITIONAL INFORMATION ON CONCENTRATIONS TESTED:
First experiment
In the absence of S9-mix, the dose levels of 1000 to 1400 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. In the presence of S9-mix, the dose levels of 1400 to 2320 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 1, 3, 10, 33, 100, 560 and 850 μg/ml exposure medium.
With S9-mix: 3, 10, 33, 100, 333 and 1000 μg/ml exposure medium.

Second experiment
In the absence of S9-mix, the dose levels of 8 to 12 μg/ml showed similar cell growth delay. Therefore, the dose level of 8 µg/ml was not regarded relevant for mutation frequency measurement. The dose levels of 15 to 28 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
In the presence of S9-mix, the dose levels of 93 to 651 μg/ml showed no cytotoxicity. Therefore, the dose levels of 93 and 279 µg/ml were not regarded relevant for mutation frequency measurement. The dose levels of 744 to 1116 μg/ml showed similar cell growth delay. Therefore, the dose levels of 744 and 930 µg/ml were not regarded relevant for mutation frequency measurement.
The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: : 0.1, 0.3, 0.6, 1, 3, 5, 9 and 12 µg/ml exposure medium.
With S9-mix: 372, 465, 558, 651, 837, 1023, 1116 and 1395 μg/ml exposure medium.

Third experiment
In the absence of S9-mix, the dose levels of 3 to 33 μg/ml showed no cytotoxicity. Therefore, the dose level of 3 µg/ml was not regarded relevant for mutation frequency measurement. The dose levels of 400 to 650 μg/ml and 700 to 900 µg/ml showed similar cell growth delay. Therefore, the dose levels of 500, 550, 800 and 900 µg/ml were not regarded relevant for mutation frequency measurement.
In the presence of S9-mix, the dose levels of 100 to 600 μg/ml showed no cytotoxicity. Therefore, the dose levels of 100 µg/ml was not regarded relevant for mutation frequency measurement. The dose levels of 800 to 1200 μg/ml showed similar cell growth delay. Therefore, the dose levels of 900 and 1000 µg/ml were not regarded relevant for mutation frequency measurement. The dose levels of 1500 and 1600 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 10, 33, 100, 300, 400, 600, 650 and 700 μg/ml exposure medium.
With S9-mix: 333, 600, 700, 800, 1100, 1200, 1300 and 1400 μg/ml exposure medium

Experiment 1: Cytotoxic and Mutagenic Response of S-10793 in the Mouse Lymphoma Test System

 Dose (µg/ml)  RSG (%) CE day2 (%)  RS day 2 (%)  RTG (%)    Mutation Frequency per 10-6 Survivors        
             Total Small   Large
Without Metabolic Activation: 3 Hours Treatment
SC1  100   127 100  100    58  ( 22  34 
SC2  100  113 100  100    55   ( 20  33 
 1  106 108  90  96    57   (  21 35 
93 99 83  77    77   ( 31  43 
10 68  118 98  66    61  (  25 33 
 33 72  127  106 76    60   (  28 30  )
 100 58  116  97 56    66   (  34 29 
560 26   120  100 26    133   (  78 45 
850  116  97   246   (  144 70 
MMS  67   91  76 51    535   (  303 171 
With Metabolic Activation: 3 Hours Treatment
SC1  100 127   100 100    77  ( 20  54 
 SC2  100 99   100 100    102   36 61 
 3  102 102  90  92    76   23 50 
10  110  88 77  85    101  (  37 60   )
 33  121 93  82  99    90  (  46 41   )
 100  96 104  92  88    88   39 45  )
 333  97 97  85  83    82   28 51   )
1000   10 89  78    252   98 126  )
 CP  49 61  54  27    1104  351  577  )
Note: All calculations were made without rounding off                                
   RSG = Relative Suspension Growth; CE = Cloning Efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent Control = Ethanol; MMS = Methylmethanesulfonate; CP = Cyclophosphamide                            

Experiment 2: Cytotoxic and Mutagenic Response of S-10793 in the Mouse Lymphoma L5178Y Test System

 Dose(µg/ml)  RSG(%)  CE day2(%)  RS day 2(%)  RTG (%)    Mutation Frequency per10-6 Survivors        
             Total Small   Large
                               Without Metabolic Activation: 24 Hours Treatment
SC1  100   123 100  100    130  ( 47  74 
SC2  100  129 100  100    103  ( 34  63 
 0.1  118 110 87  103   84  (  20 61 
0.3  93  127 101  94    82   ( 17  62 
0.6 89   102 81 73    79  (  20 57 
 1 114   118  94 106   75   (  22 51  )
 3 152   111  88 135   93   (  28 61 
5 51   133 105  53   96   ( 24 68 
9 15  105  84 12   84   (  23 58
12 14   143  113 16    68  (  18 48
 MMS  81  75  59  48   1363   (  427 610  )
With Metabolic Activation: 3 Hours Treatment   
 SC1  100 98  100 100    129  31 92 
 SC2  100 78 100 100   125  41 78 
372 112  89 101  113    123 (  31 86   )
 465 104 79  90  94    148 (  31 110   )
 558  80 89  101  80   211  59 135  )
 651  84 111 127 107   111  31 74   )
837  67 110 125 83   186  43 128  )
 1023  63 91 104 66   160 53  97  )
 1116 57  105  120  68    129   25 97  )
 1395 29  77  87  25    252   54 180     )    
 CP  60 55  62  37    1284  319  772 
Note: All calculations were made without rounding off                    
RSG = Relative Suspension Growth; CE = Cloning Efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent Control = Ethanol; MMS = Methylmethanesulfonate; CP = Cyclophosphamide                     

Experiment 3: Cytotoxic and Mutagenic Response of S-10793 in the Mouse Lymphoma L5178Y Test System

Dose(µg/ml)  RSG(%)  CE day2(%)  RS day 2(%)  RTG (%)    Mutation Frequency per10-6 Survivors        
             Total Small   Large
                               Without Metabolic Activation: 24 Hours Treatment
SC1  100   75 100  100    59  ( 41  16 
SC2  100  79 100  100    68  ( 48  19 
 10 109 94 91  99   70  ( 46 22 
33  90  83 96  86    49   ( 29  19 
100 72   83 99 71    39  ( 19 19 
 300 58   63  91 53   48   (  32 15  )
400 48   59  81 38   70   (  46 22 
600 47   46 87  41   55   ( 26 28 
650 34  30 91 31   61   (  29 30
700 17   11  66 11   95  (  45 47
 MMS 76  45  57  43   654   (  450 155  )
With Metabolic Activation: 3 Hours Treatment   
 SC1  100 75  100 100   72  45 26 
 SC2  100 79 100 100   80  46 31 
333 96 94 122  118    52 (  31 20   )
 600 81 83  107  87   71 (  36 32   )
 700 79 83  107  85   72  38 32  )
 800 63 63 82 52   96  49 44   )
1100 59 71 93 55   71  48 21  )
 1200 46 81 106 49   75 51 22  )
 1300 30 81  106  32    67 46 19  )
 1400 11 65  85    153  123 26     )    
 CP 45 52  68  30    757 463 227 
Note: All calculations were made without rounding off                    
RSG = Relative Suspension Growth; CE = Cloning Efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent Control = Ethanol; MMS = Methylmethanesulfonate; CP = Cyclophosphamide                     
Conclusions:
Taken together, it is concluded that the mutagenic response observed in the mouse lymphoma L5178Y test system is negative. Increases in the mutation frequency at the TK locus are only observed in the first experiment, and occur at severely cytotoxic concentrations that exceed the OECD guidance stating that the RTG should not go below 10%, since survival rates below 10% give rise to false positives. The mutation frequencies above the GEF + MF(controls) in the absence and presence of S9-mix were not confirmed in the second and third mutation experiment.
Executive summary:

Evaluation of the mutagenic activity of the test material in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (with independent repeat) was done according to OECD TG 476 and under GLP conditions

* In the first experiment, the test material was tested up to concentrations of 850 and 1000 µg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. It was tested up to cytotoxic levels of 93 and 92% in the absence and presence of S9-mix, respectively.

 

* In the second experiment, the test material was tested up to concentrations of 12 and 1395 µg/ml in the absence and presence of S9-mix, respectively. The incubation times were 24 hours for incubations in the absence of S9-mix and 3 hours for incubations in the presence of S9-mix. It was tested up to cytotoxic levels of 84 and 75% in the absence and presence of S9-mix, respectively

 

* Since no clear conclusion about the mutagenicity could be made, an additional third mutation experiment was performed. The test matreial was tested up to concentrations of 700 and 1400 µg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. It was tested up to cytotoxic levels of 89 and 91% in the absence and presence of S9-mix, respectively.

 

It was confirmed that the spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Mutation frequencies in cultures treated with positive control chemicals were increased by 9.5-, 12- and 11-fold for MCM in the absence of S9-mix, and by 12- and 10-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of

S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

 

In the absence of S9-mix, in the first experiment, the test matreial induced an up to 4.4-fold dose-related increase in mutation frequency (at concentrations of 560 µg/ml and higher) and a mutation frequency above the GEF + MF(controls)of 246 x 10-6(at 850 µg/ml) as well as above the laboratory’s historical solvent control range. This response fulfilled the criteria for a positive response.The increase above the GEF + MF(controls)was only observed at the highest tested concentration of 850 µg/ml with severe toxicity (relative total growth: RTG of 7%). The next lower dose levels of 100 and 560 μg/ml showed toxicity of 56 and 26%, respectively. At these concentrations no biologically significant increase were observed in the mutation frequency at the TK locus. This increase could not be repeated in the additional experiment in which S-10793 was tested up to a dose level with a RTG of 89%. In the experiment with a prolonged treatment period, the test material did not induce an increase in mutation frequency up to dose levels with a RTG of 12 and 16%.

 

In the presence of S9-mix, the test material induced up to 2.8- and 2.0-fold increases in mutation frequency in the first and second experiment, respectively. The increases were above the laboratory’s historical solvent control range in both experiments. The increases observed were above the GEF + MF(controls) (216 per 106survivors) in the first experiment and just below the GEF + MF(controls)(253 per 106survivors) in the second experiment. However the increase observed in the first experiment was only observed at the top concentration of 1000 µg/ml with a RTG of 8% and the increase observed in the second experiment was not above the GEF + MF(controls) and mutation frequency of the next lower dose level of 1116 μg/ml was comparable to the solvent controls. In the repeat experiment, the test material was tested up to a dose level with a RTG of 91% and no dose level was observed with an increase in the mutation frequency of more than 2-fold or above the GEF + MF(controls).

Taken together, it is concluded that the mutagenic response observed in the mouse lymphoma L5178Y test system is negative. Increases in the mutation frequency at the TK locus are only observed in the first experiment, and occur at severely cytotoxic concentrations  that exceed the OECD guidance stating that the RTG should not go below 10%, since survival rates below 10% give rise to false positives. The mutation frequencies above the GEF + MF(controls) in the absence and presence of S9-mix were not confirmed in the second and third mutation experiment.

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

Additional information

Additional information from genetic toxicity in vitro:

Regarding the MLA study:

The authors summarize as the study design and results as follows. In the first experiment, S-10793 was tested up to concentrations of 850 and 1000 µg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. S-10793 was tested up to cytotoxic levels of 93 and 92% in the absence and presence of S9-mix, respectively.

In the second experiment, S-10793 was tested up to concentrations of 12 and 1395 µg/ml in the absence and presence of S9-mix, respectively. The incubation times were 24 hours for incubations in the absence of S9-mix and 3 hours for incubations in the presence of S9-mix. S-10793 was tested up to cytotoxic levels of 84 and 75% in the absence and presence of S9-mix, respectively

 

Since no clear conclusion about the mutagenicity of S-10793 could be made, an additional third mutation experiment was performed. S-10793 was tested up to concentrations of 700 and 1400 µg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. S-10793 was tested up to cytotoxic levels of 89 and 91% in the absence and presence of S9-mix, respectively.

 

It was confirmed that the spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Mutation frequencies in cultures treated with positive control chemicals were increased by 9.5-, 12- and 11-fold forin the absence of S9-mix, and by 12- and 10-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of

S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

 

In the absence of S9-mix, in the first experiment, S-10793 induced an up to 4.4-fold dose-related increase in mutation frequency (at concentrations of 560 µg/ml and higher) and a mutation frequency above the GEF + MF(controls)of 246 x 10-6(at 850 µg/ml) as well as above the laboratory’s historical solvent control range. This response fulfilled the criteria for a positive response.The increase above the GEF + MF(controls)was only observed at the highest tested concentration of 850 µg/ml with severe toxicity (relative total growth: RTG of 7%). The next lower dose levels of 100 and 560 μg/ml showed toxicity of 56 and 26%, respectively. At these concentrations no biologically significant increase were observed in the mutation frequency at the TK locus. This increase could not be repeated in the additional experiment in which S-10793 was tested up to a dose level with a RTG of 89%. In the experiment with a prolonged treatment period, S-10793 did not induce an increase in mutation frequency up to dose levels with a RTG of 12 and 16%.

 

In the presence of S9-mix, S-10793 induced up to 2.8- and 2.0-fold increases in mutation frequency in the first and second experiment, respectively. The increases were above the laboratory’s historical solvent control range in both experiments. The increases observed were above the GEF + MF(controls)(216 per 106survivors) in the first experiment and just below the GEF + MF(controls)(253 per 106survivors) in the second experiment. However the increase observed in the first experiment was only observed at the top concentration of 1000 µg/ml with a RTG of 8% and the increase observed in the second experiment was not above the GEF + MF(controls)and mutation frequency of the next lower dose level of 1116 μg/ml was comparable to the solvent controls. In the repeat experiment, S-10793 was tested up to a dose level with a RTG of 91% and no dose level was observed with an increase in the mutation frequency of more than 2-fold or above the GEF + MF(controls).

Ames test results were supportive of the MLA study result:

S-10793 did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

This result is also supported by an in vitro mutagenicity study using human lymphocytes (NOTOX 2012). S-10793 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments. No effects of S-10793 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. The study observations support the conclusion that S-10793 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in the study.

Justification for classification or non-classification

Not Classified. - Based on available data and/or professional judgment, the classification criteria are not met.