Rhamnolipids: fermentation products of glucose with Pseudomonas putida, Pseudomonadaceae

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The experimental data on the in vitro genetic toxicity in bacterial and mammalian cells for the target substance Rhamnolipids: fermentation products of glucose with Pseudomonas bacteria did not show any effect on mutagenicity

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-07-24 to 2015-01-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 22 July, 2010

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light and humidity
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: soluble in water

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
taking into account the sample composition of 79% by applying a correction factor of 1.26, the
test item was dissolved in the vehicle
- Final dilution of a dissolved solid, stock liquid or gel:
100 mg/mL for the preliminary toxicity test,
8 mg/mL for the first experiment with S9 mix,
6 mg/mL for the first experiment without S9 mix and the second experiment with S9 mix,
2 mg/mL for the second experiment without S9 mix.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ATCC (American Type Culture Collection, Manassas, USA)
- Suitability of cells: cell line recommended by international regulations for in vitro mammalian cell gene mutation test and for in vitro micronucleus test.
- Cell cycle length, doubling time or proliferation index: average cell cycle time approximately 10-12 hours

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 μg/mL) and sodium pyruvate (200 μg/mL). Medium was supplemented by heat-inactivated horse serum at 10% (v/v) and 5% of S9 mix (tests with metabolic activation).
Media during treatment: culture medium containing 5% inactivated horse serum, +- test item +- S9 mix
Media during recovery period: culture medium containing 10% inactivated horse serum
- atmosphere of 5% CO2/95% air
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no information
- Periodically 'cleansed' against high spontaneous background: no information

Additional strain / cell type characteristics:

other: thymidine kinase deficient

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from liver of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

preliminary test: 10, 100, 500, 1000, 2500 and 5000 μg/mL

Justification for top dose:
Following the short treatment periods with and without S9 mix, a severe toxicity was observed at dose-levels> 500 μg/mL, as shown bya 100% decrease in the PD.Following the 24-hour treatment, a severe toxicity was observed at dose-levels 2> 100 μg/mL, as shown by a 100% decrease in the PD.

Experiments without S9 mix
12.5, 25, 50, 100, 125, 150,200 and 300 μg/mL for the 3 h treatment,
1.56, 3.13, 6.25, 12.5, 25, 37.5, 50 and 100 μg/mL for the 24 h treatment.

Experiments with S9 mix
12.5, 25, 50, 100, 150, 200, 300 and 400 μg/mL for the first experiment,
12.5, 25, 50, 100, 125, 150, 200 and 300 μg/mL for the second experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: available solubility data
Pretest:
At the highest dose-level of 5000 μg/mL, the pH was approximately 7.4 (as for the vehicle control) and the osmolality was equal to 298 mOsm/kg H20 (286 mOsm/kg for the vehicle control).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Colchicine without S9 mix: 3h treatment (0.5 µg/mL) and 24 h treatment (0.5 µg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Without S9 mix first exp: 3 h treatment + 24 h recovery; second exp: 24 h treatment + 20 h recovery
With S9 mix first exp: 3 h treatment + 24 h recovery; second exp: 3 h treatment + 24 h recovery

NUMBER OF REPLICATIONS: two independent experiments, two cultures/dose-Ievel

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Cells were washed with culture medium containing 10% inactivated horse serum and 1% pluronic acid.
The cells were suspended in 49.5% culture medium containing 10% inactivated horse serum, 50% PBS and 0.5% pluronic acid, before being fixed.
Following the fixation, the cells were kept at 4°C for at least an overnight period.

Slides were air-dried before being stained for approximately 15 min in 5% Giemsa


NUMBER OF CELLS EVALUATED: 1000 mononucleated cells per culture

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
according to recommendationsof Miller et al. (1995):
- micronuclei should be clearly surrounded by a nuclear membrane,
- the micronucleus area should be less than one-third of the area of the main nucleus,
- non-refractility of the micronuclei,
-micronuclei should not be linked to the main nucleus via nucleoplasmic bridges,
- micronuclei should be located within the cytoplasma of the cell,
- only mononucleated cells with a number of micronuclei ~ 5 should be scored to exclude apoptosis and nuclear fragmentation.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (Decrease in PD (%) = 100 - Mean PD as % of control)

Rationale for test conditions:

Micronucleus analysis
The dose-levels selected for micronucleus analysis were as follows:
50, 100, 150 μg/mL for the 3-hour treatment, the latter being the lowest dose-level with emulsion and higher dose-levels being too cytotoxic,
25,37.5, 50 μg/mL for the 24-hour treatment, the higher tested dose-level being too cytotoxic.

Evaluation criteria:

Evaluation of a positive response: a test item is considered to have castogenic and/or aneugenic potential, if all the following criteria were met:
- a dose-related increase in the frequency of micronucleated cells was observed, for at least one dose-level,
- the frequency of micronucleated cells of each replicate culture was above the corresponding vehicle historical range,
- a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose-levels.

Evaluation of a negative response:
- a test item is considered negative if none of the criteria for a positive response were met.

Statistics:

For each experiment, the frequency of micronucleated cells in treated cultures was compared to that of the vehicle control cultures.
This comparison was performed using the X2 test, unless treated culture data are lower than or equal to the vehicle control data. P = 0.05 was used as the lowest level of significance.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

3-hour treatment: severe toxicity (100% decrease in the PD) at dose-levels ≥ 200 μg/mL 24-hour treatment: severe toxicity (100% decrease in the PD) at the highest dose-level of 100 μg/mL

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: at highest dose-level of 5000 μg/mL, the pH was approximately 7.4 (as for the vehicle control)
- Effects of osmolality: osmolality was equal to 298 mOsm/kg H20 (286 mOsm/kg for the vehicle control)
- Evaporation from medium: not expected
- Water solubility: established in pretest
- Precipitation: A slight emulsion was observed at the end of the treatment period at dose-levels ≤ 150 μg/mL.

RANGE-FINDING/SCREENING STUDIES:
10, 100, 500, 1000, 2500 and 5000 μg/mL.
3 h exposure with and without S9: severe toxicity (100% decrease in the PD) was observed at dose-levels ≥ 500 μg/mL
24-h-treatment: severe toxicity (100% decrease in the PD) was observed at dose-levels ≥ 100 μg/mL


NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: 1000


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
MMC
without S9 3 hours treatment + 24 hours recovery:
n= 38; mean= 142.4; SD= 74.2; lower CL95%= 118.0; upper CL95%= 166.8; 95th percentile= 262.5
without S9 24 hours treatment + 20 hours recovery:
n= 40; mean= 85.5; SD= 51.4; lower CL95%= 69.1; upper CL95%= 101.9; 95th percentile= 191.5

COL
without S9 3 hours treatment + 24 hours recovery:
n= 39; mean= 28.8; SD= 15.5; lower CL95%= 23.8; upper CL95%= 33.8; 95th percentile= 68.0
without S9 24 hours treatment + 20 hours recovery:
n= 41; mean= 36.8; SD= 12.3; lower CL95%= 32.9; upper CL95%= 40.7; 95th percentile= 55.0

CPA
with S9 3 hours treatment + 24 hours recovery
n= 72; mean= 96.8; SD= 43.7; lower CL95%= 86.5; upper CL95%= 107.0; 95th percentile= 165.5


- Negative (solvent/vehicle) historical control data:
without S9 3 hours treatment + 24 hours recovery:
n= 39; mean= 1.9; SD= 1.2; lower CL95%= 1.6; upper CL95%= 2.3; 95th percentile= 4.5
without S9 24 hours treatment + 20 hours recovery:
n= 41; mean= 2.0; SD= 1.3; lower CL95%= 1.6; upper CL95%= 2.4; 95th percentile= 4.5
with S9 3 hours treatment + 24 hours recovery:
n= 72; mean= 1.8; SD= 1.2; lower CL95%= 1.5; upper CL95%= 2.0; 95th percentile= 4.0



ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RPD
- Other observations when applicable:
Micranucleus analysis
The dose-levels selected for micronucleus analysis were as follows:
50, 100, 150 μg/mL for the 3-hour treatment, the latter being the lowest dose-level with emulsion and higher dose-levels being too cytotoxic,
25,37.5, 50 μg/mL for the 24-hour treatment, the higher tested dose-level being too cytotoxic.

First experiment without S9 mix (3-h treatment + 24-h recovery), mutagenicity

 

Treatment	Mean PD as % of control	Total micronucleated cells per dose	Frequency of micronucleated cells ‰	Ratio treated/control
Vehicle control	100	4	2
Test item (µg/mL)
50	102		4	1.0
100	89		6	1.5
150 E	75		9	2.3
Positive controls
MMC (1 μg/mL)	#		107	53.5***
COL (0.5 μg/mL)	#		32	16.0***

 

Second experiment without S9 mix (24-h treatment + 20-h recovery), mutagenicity

 

Treatment	Mean PD as % of control	Total micronucleated cells per dose	Frequency of micronucleated cells ‰	Ratio treated/control
Vehicle control	100	2	1
Test item (µg/mL)
25	92	1	1	0.5
37.5	99	4	2	2.0
50	89	5	3	2.5
Positive controls
MMC (0.1 μg/mL)	92	37	19	18.5***
COL (0.5 μg/mL)	#	108	54	54.0***

(1): expressed as active item                                                   Statistics: 2 x 2 contingency tabie:

PD: population doubling                                                            ••• : p< 0.001

Vehicle control: water for injections, MMC: Mitomycin C, COL: Colchicine

#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment

E: emulsion was noted in the culture medium at the end of treatment

First experiment with S9 mix (3-h treatment + 24-h recovery), mutagenicity

 

Treatment	Mean PD as % of control	Total micronucleated cells per dose	Frequency of micronucleated cells ‰	Ratio treated/control
Vehicle control	100	3	2
Test item (µg/mL)
50	107	10	5	3.3
100	119	4	2	1.3
150 E	121	5	3	1.7
Positive controls
CPA (6 μg/mL)	73	136	68	45.3***

(1): expressed as active item                                                   Statistics: 2 x 2 contingency tabie:

PD: population doubling                                                            ••• : p< 0.001

Vehicle control: water for injections, CPA: Cyclophosphamide

E: emulsion was noted in the culture medium at the end of treatment

 

Second experiment with S9 mix (3-h treatment + 24-h recovery), mutagenicity

Treatment	Mean PD as % of control	Total micronucleated cells per dose	Frequency of micronucleated cells ‰	Ratio treated/control
Vehicle control	100	6	3
Test item (µg/mL)
50	106	1	1	0.2
100	115	4	2	0.7
150 E	133	7	4	1.2
Positive controls
CPA (6 μg/mL)	22	171	86	28.5***

(1): expressed as active item                                                   Statistics: 2 x 2 contingency tabie:

PD: population doubling                                                            ••• : p< 0.001

Vehicle control: water for injections, CPA: Cyclophosphamide

Conclusions:

Under the experimental conditions of the study, the test item, did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+- mouse lymphoma cells, in the absence or in the presence of a rat metabolising system.

Executive summary:

In an In Vitro Mammalian Cell Micronucleus Test according to OECD guideline 487, L5178Y TK^± mouse lymphoma cells cultured in vitro were exposed to the test substance Rhamnolipids at the following concentrations in the presence and absence of mammalian metabolic activation (S9- mix of Arochlor 1254 induced rat liver).

Preliminary Toxicity Assay (with and without S9 mix): 10, 100, 500, 1000, 2500 and 5000 µg/mL.

Severe toxicity (100% decrease in the PD) was observed following the short treatment periods with and without S9 mix, at dose-levels ≥ 500 μg/mL and following the 24-hour treatment at dose-levels ≥ 100 μg/mL.

Experiments without S9 mix

12.5, 25, 50, 100, 125, 150, 200 and 300 μg/mL for the 3 h treatment,

1.56, 3.13, 6.25, 12.5, 25, 37.5, 50 and 100 μg/mL for the 24 h treatment.

 

Experiments with S9 mix

12.5, 25, 50,100,150, 200, 300 and 400 μg/mL for the first experiment,

12.5, 25, 50, 100, 125, 150, 200 and 300 μg/mL for the second experiment.

 

Water was selected as solvent and treatment volume for the testsustance was 5% (v/v) in culture medium. A slight emulsion was observed at the end of the treatment periods at dose-levels ≥150 μg/mL.

 

In both experiments with S9 mix, a severe toxicity was induced at dose-levels ≥ 300 μg/mL, as shown by a 100% decrease in the PD. In the experiments without S9 mix, following the 3-hour treatment, severe toxicity was induced at dose-levels≥ 200 μg/m L and following the 24-hour treatment at the highest dose-level of 100 μg/mL.

 

The dose-levels selected for micronucleus analysis were as follows:

50, 100, 150 μg/mL for experiments with S9 mix and the 3-hour treatment without S9 mix and 25, 37.5, 50 μg/mL for the 24-hour treatment without S9 mix.

 

No significant increase in the frequency of micronucleated cells was noted in either experiment after the 3-hour treatment with or without S9 mix and after the 24-hour treatment without S9 mix.

 

The positive controls did induce the appropriate response. 

 

The results of the in vitro Mammalian Cell Micronucleus Test indicate that, the test substance, Rhamnolipids did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK^± mouse lymphoma cells, in the absence or in the presence of a rat metabolising system and was concluded to be negative under the conditions of this study.

 

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

Adopted 29 July 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: ET19404365, Source: Sponsor
- Purity, including information on contaminants, isomers, etc.: 49.2% (liquid aqueous solution
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (+14°C ÷ +25°C), protected from light

Target gene:

HPRT gene

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Remarks:

V79 fibroblast colonies

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Chinese hamster V79 cells, are derived from a culture of embryonic lung tissue of male Chinese hamster (Cricetulus griseus). Chinese hamster V79 cells were obtained from Dr. J. Thacker, MRC Radiobiology Unit, Harwell, UK.
- Suitability of cells: The use of the HPRT mutation system in Chinese hamster V79 cells has been well characterized and validated and is accepted by many regulatory authorities.

For cell lines:
- Absence of Mycoplasma contamination: The cells are checked at regular intervals for the absence of mycoplasmal contamination
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes
The karyotype, generation time, plating efficiency and mutation rates (spontaneous and induced) have been checked in this laboratory
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
The following culture media were used:
-EMEM Minimal medium:
Eagle’sMinimal EssentialMedium (10X) 58.7mL
L-glutamine (200 mM) 5.9mL
Sodium bicarbonate (7.5%) 15.7mL
Non-essential amino acids (100X) 5.9mL
Streptomycin sulphate 50000 IU/mL +
Penicillin G 50000 IU/mL 1.2mL
Sterile bidistilled water 500mL

-EMEM Complete medium (10%)
EMEM Minimal medium 900mL
Foetal Calf Serum 100mL
At Day 0 (determination of survival): At the end of treatment, the medium was removed and the cell monolayers were washed with PBS. The cultures were trypsinised, counted and an aliquot from each culture was diluted and plated to estimate the viability of the cells. Each cell suspension was re-plated (2 x 106cells/ F175) in order to maintain the treated cell populations. Fresh complete medium was added to the flasks which were then returned to the incubator at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.

Metabolic activation:

with and without

Metabolic activation system:

S9 Homogenate
One batch of S9 tissue fraction used had following characteristics:
Species: Rat
Strain: Sprague Dawley
Tissue: Liver
Inducing Agents: Phenobarbital – 5,6-Benzoflavone
Producer: MOLTOX, Molecular Toxicology, Inc.
Batch Number:3971
The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared as follows (for each10 mL):
S9 tissue fraction: 2.0mL
NADP (0.1 M): 0.4mL
G-6-P (0.1 M): 0.5mL
KCl (0.33 M): 1.0mL
MgCl2 (0.1M). 0.5mL
Phosphate Buffer (0.2 M): 5.6mL

Test concentrations with justification for top dose:

Cytotoxicity assay: A preliminary cytotoxicity test was undertaken in order to select appropriate dose levels for the mutation assay. The test item was assayed at a maximum dose level of 1910 μg/mL and at a wide range
of lower dose levels: 954, 477, 239, 119, 59.7, 29.8, 14.9 and 7.46 μg/mL. In the absence of
S9 metabolism, no cells survived treatment starting from 239 μg/mL onwards; while no
reduction of Relative Survival (RS) was noticed over the remaining concentrations tested.
In the presence of S9 metabolism, no cells survived treatment at the two highest dose
levels (1910 and 954 μg/mL), while no reduction of RS was observed over the remaining
concentrations tested. No precipitation was noted at any concentration tested.

Mutation assays: Based on the results obtained in the preliminary cytotoxicity test, a Main Assay was performed using tjhe following dose levels:
with S9 : 229, 183, 147, 117, 93.9 and 75.1 µg/mL
without S9: 858, 686, 549, 439, 351 and 281 µg/mL

Vehicle / solvent:

The solvent used in this study was EMEM MinimalMedium (ERBC, batch nos: 005/331 and 005/332).
Solutions of ethylmethanesulfonate (Sigma, batch no.: BCBV9352) were prepared in ethanol (C. Erba, batch no.: V7N479127N) and served as positive controls in the absence of S9 metabolism. Solutions of 7,12-dimethylbenzanthracene (labelled as 7,12-dimethylbenz(a)anthracene; Sigma, batch no.: SLBF3276V) were prepared in DMSO (Honeywell, batch no.: H0795) and served as positive control in the presence of S9 metabolism.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate cultures
- Number of independent experiments: Main Assay: Two treatment series assayed in separate runs
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Culture were incubated at 37°C for three hours
- Harvest time after the end of treatment (sampling/recovery times): Day0: At the end of treatment, the medium was removed and the cell monolayers were washed with PBS. The cultures were trypsinised, counted and an aliquot from each culture was diluted and plated to estimate the viability of the cells. Each cell suspension was re-plated (2 x 106cells/ F175) in order to maintain the treated cell populations. Fresh complete medium was added to the flasks which were then returned to the incubator at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.
Day 2 and 5 (subculturing): On Days 2 and 5, the cell populations were subcultured in order to maintain them in exponential growth. The cultures were trypsinised, counted and the number of cells taken forward was adjusted to give two million viable cells seeded in 225 cm2 flasks
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): At the expression time (Day 8), each culture was trypsinised, resuspended in complete
medium and counted by microscope. After dilution, an estimated 1 x 105 cells were plated in each of twenty 100mm tissue culture petri dishes containing medium supplemented
with 6-thioguanine (at 7.5 μg/mL). These plates were subsequently stained with Giemsa solutions and scored for the presence of mutants. After dilution, an estimated 200 cells
were plated in each of three 60mmtissue culture petri dishes. These plates were used to estimate Cloning Efficiency (CE).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g: Cytotoxicity was evaluated by relative survival (RS), i.e. cloning efficiency (CE) of cells plated immediately after treatment, adjusted by any loss of cells during treatment, as compared with adjusted cloning efficiency in solvent controls (assigned a survival of 100%).

Rationale for test conditions:

The gene mutation assay method used here is based on the identification of V79 fibroblast
colonies which have become resistant to a toxic purine analogue (6-thioguanine). This analogue
can be metabolised by the enzyme hypoxanthine-guaninphosphoribosyl-transferase
(HPRT) into nucleotides, which are used in nucleic acid synthesis resulting in the death of
HPRT-competent cells. HPRT-deficient cells, which are presumed to arise through mutations
in the HPRT gene, cannot metabolise 6-thioguanine and thus survive and grow in
its presence. The mutations induced are recessive. However, since the gene which codes
for the HPRT enzyme is located on the X chromosome, of which only one copy is present
in male cells, a single mutation is sufficient for the mutant genotype to be observed. The
cells used, Chinese hamster V79 cells, are derived from a culture of embryonic lung tissue
of male Chinese hamster (Cricetulus griseus). The use of the HPRT mutation system in
Chinese hamster V79 cells has been well characterised and validated and is accepted by
many regulatory authorities.

Evaluation criteria:

ACCEPTANCE CRITERIA
The assay was considered valid if the following criteria were met:
– The mutant frequency of the solvent/vehicle control is within the 95% control limits of the distribution of the laboratory’s historical control database.
– The positive controls induce responses that are compatible with those generate in the historical control database and produce a statistically significant increase in mutant frequency, compared with the concurrent solvent/vehicle control.
– Two experimental conditions are tested (i.e. with and without metabolic activation), unless one gives positive results.
– Adequate number of cells (i.e. at least 20 x 10*6 at treatment time and at least 2 x 10*6 during the expression period) and concentrations (at least 4 with appropriate cytotoxicity) are analysable.
– The selection of dose levels is consistent with those indicated in Test concentration section of the present RSS.

CRITERIA OF THE OUTCOME ASSAY
A test item is considered to be clearly positive if:
– At least one of the test concentrations exhibits a statistically significant increase, compared with the concurrent solvent/vehicle control.
– The increase is concentration-related.
– Any of the results are outside the distribution of the historical negative control data (95% confidence limits).

A test item is considered to be clearly negative if:
– None of the test concentrations exhibits a statistically significant increase, compared
with the concurrent solvent/vehicle control.
– There is no concentration-related increase.
– All results are inside the distribution of the historical negative control data (95%
confidence limits).
Historical control data are used to demonstrate biological relevance of the results obtained

Statistics:

The individual mutation frequency values at each test point were transformed to induce
homogeneous variance and normal distribution. The appropriate transformation was
estimated using the procedure of Snee and Irr (1981), and was found to be y = (x +a)b
where a = 0 and b = 0.275.
The mutant frequency in the solvent control and treated cultures was compared using the
Dunnett’s test (one-tailed).
For each experimental point, the corrected sum of squares of transformed mutation frequencies.
The error mean square (EMS) was calculated as the sum of SSy values divided by the sum
of degrees of freedom.
For each comparison of treatment with control, the calculated t value was compared with
tabulated critical values for the one tailed Dunnett’s test.
The results of the experiment were subjected to an Analysis of Variance in which the effect
of replicate culture and dose level in explaining the observed variation were examined. For
each experiment, a two way analysis of variance was performed (without interaction) fitting
to two factors:
– Replicate culture: to identify differences between the replicate cultures treated.
– Dose level: to identify dose-related increases (or decreases) in response, after allowing
for the effects of replicate cultures and expression time.
The analysis was performed separately with the sets of data obtained in the absence and
presence of S9 metabolism.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the absence and presnceof S9 metabolism, no cell survived treatment at the highest concentration tested

Vehicle controls validity:

valid

Positive controls validity:

valid

EXPERIMENTAL DESIGN

Based on the results obtained in the preliminary cytotoxicity test, a Main Assay for mutation to 6-thioguanine resistance was performed using dose levels :

1st assay,  – S9:  229, 183, 147, 117, 93.9 and 75.1 µg/mL

1nd assay, + S9: 858, 686, 549, 439, 351 and 281 µg/mL

The mutant frequencies in the negative control cultures fell within the 95% control limits of the distribution of the laboratory’s historical negative control data. Treatment with the

positive control items gave marked responses that were compatible with those generated in the historical control data base and produced a statistically significant increase in mutant

frequency, compared with the concurrent solvent/vehicle control, indicating the correct functioning of the test system. Adequate number of cells and concentrations was analysed.

The study was accepted as valid.

OSMOLALITY AND pH

The pH values and osmolality of the post-treatment media (see Attached background Information). The addition of the test item solution did not have any obvious effect on the osmolality or pH of the treatment medium.

SURVIVAL AFTER TREATMENT

In the absence of S9 metabolism, no cell survived treatment at the highest concentration tested. Marked toxicity was observed at the next lower concentration of 183 μg/mL, reducing

RS to 10% of the negative control, while moderate toxicity (RS=23%) was noticed at 147 μg/mL. Mild to no toxicity was seen over the remaining concentrations tested. In the

presence of S9 metabolic activation, no cell survived treatment at the highest dose level, while a slight reduction in RS (82%) was seen at the next lower concentration of 686 μg/mL.

Due to the steep decline of survival, no concentration tested in this treatment series showed an adequate toxicity (20-10% RS). Since a very narrow spacing between dose levels (1.25

dilution factor) was used, the study was nevertheless accepted as valid. No precipitation was noted at any concentration tested.

MUTATION RESULTS

No statistically significant increases over the spontaneous mutation frequency were observed at any treatment level in the absence or presence of S9 metabolic activation.

Results of the mutation frequency at all concentrations tested were within the distribution of the historical negative control data.

Analysis of variance indicated that dose levels and replicate cultures were not significant factors in explaining the observed variation in the data, in the absence or presence of S9

metabolism.

Although no concentration tested in the presence of S9 metabolism showed adequate toxicity to assess mutation induction, data obtained were considered adequate to demonstrate

a clear evidence of negative results

Conclusions:

It is concluded that the test item does not induce mutation in Chinese hamster V79 cells
after in vitro treatment, in the absence or presence of S9 metabolic activation, under the
reported experimental conditions

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-06-23 to 2014-07-15

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:

adopted 21st July, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

dated May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Solubility and stability of the test substance in the solvent/vehicle: no precipitation of the test item at any concentration used


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: dissolved in A. dest.



FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS:

Target gene:

Histidine locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

ral liver S9 mix

Test concentrations with justification for top dose:

range finding study: TA98 and TA100 with and without S9: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
Experiment I: plate incorporation
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
Experiment II: pre-incubation
10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate (forTA 98, TA 1535, TA 1537 and TA 102)
1.00, 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate (for TA 100)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: solubility pretest

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (without metabolic activation); 2-aminoanthracene (with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation; preincubation)

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h


NUMBER OF REPLICATIONS: 3 (test substance, positive controls), 10 negative controls in 2 independent assays

DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately s 0.5 in relation to the solvent control.

Evaluation criteria:

A test item is considered as mutagenic if:
a clear and dose-related increase in the number of revertants occurs and/or a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as folIows:
if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: given as established in pretest
- Precipitation: No precipitation of the test item was observed in any tester strain used in experiment land II (with and without metabolic activation).
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES:
A range finding study was conducted in tester strains TA98 and TA100

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: the positive control plates with and without S9 mix were within the historical control data range
- Negative (solvent/vehicle) historical control data: the negative control plates with and without S9 mix were within the historical control data range

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Toxic effects of the test item were noted in three tester strains evaluated in experiment l and II. In experiment I toxic effects of the test item were observed in tester strains TA 100 at concentrations of 1000 μg/plate and higher (without metabolic activation) and at concentrations of 2500 μg/plate (with metabolic activation). In tester strains TA 1535 and TA 1537 toxic effects of the test item were noted at concentrations of 2500 μg/plate and higher (with and without metabolic activation).

In experiment II toxic effects of the test item were noted in tester strain TA 100 at concentrations of 100 μg/plate and higher (without metabolic activation) and at concentrations of 316 μg/plate and higher (with metabolic activation). In tester strain TA 1535 toxic effects of the test item were observed at concentrations of 1000 μg/plate and higher (without metabolic activation) and at concentrations of 2500 μg/plate and higher (with metabolic activation). In tester strain TA 1537 toxic effects of the test item were observed at concentrations of 1000 μg/plate and higher (without metabolic activation) and at a concentration of 5000 μg/plate (with metabolic activation).

- Other observations when applicable: The
reduction in the number of revertants down to a mutation factor of 0.5 found in tester strain TA 1537 at a concentration of 316 μg/plate (without metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship.

Negative as well as positive controls were included in each experiment. Strain specific positive controls were included in the assay, which demonstrated the effective performance of the test.

Negative/Solvent Controls:

Negative controls (A. dest.) were treated in the same way as all dose groups.

Positive Controls:

with S9: 2.5 μg/plate 2-aminoanthracene (TA 98, TA 100, TA 1535, TA 1537); 10 μg/plate 2-aminoanthracene for TA 102

without S9:      sodium azide 10 μg/plate for TA 100, TA 1535

4-nitro-o-phenylene-diamine 10 μg/plate for TA 98, 40 μg/plate for TA 1537

methylmethanesulfonate 1 μL/plate for TA102

Conclusions:

The test substance was evaluated in the bacterial reverse mutation assay (Ames test) using Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537 and TA102 in the presence and absence of rat liver S9 mix. Under the conditions of the study, the test substance was negative for mutagenic potential.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted 21 July 1997), Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA102 were exposed to the test substance  in water in concentrations of 0 (control), 1.00*, 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate in the absence and presence of mammalian metabolic activation (rat liver S9 mix) (*only for TA100). The assay was performed using the plate incorporation method.

The test substance was tested up to cytotoxic concentrations. Toxic effects of the test item were noted in three tester strains used in experiment l and II: In experiment I toxic effects of the test item were observed at concentrations of 1000 μg/plate and higher (without metabolic activation) and at concentrations of 2500 μg/plate and higher (with metabolic activation), depending on the particular tester strain.

In experiment II toxic effects of the test item were noted at concentrations of 100 μg/plate and higher (without metabolic activation) and at concentrations of 316 μg/plate and higher (with metabolic activation), depending on the particular tester strain.

Precipitation was not observed. The positive controls induced the appropriate responses in the corresponding strains. The mean numbers of revertant colonies in the negative controls were within the ranges of the historical control data.

There was no evidence of an increase in the number of revertant colonies that exceeded twice background in any of the five tester strains (TA98, TA 100, TA1535, TA1537 or TA102) examined at dose levels up to 5000 µg/plate in the absence or in the presence of S9. Therefore, test substance was considered to be non-genotoxic (non-mutagenic) in Salmonella tester strains TA98, TA 100, TA1535, TA1537 or TA102 under the conditions employed (plate incorporation assay and pre-incubation method).

There was no evidence of induced mutant colonies over background.

Under the conditions of the study, the test substance was negative for mutagenic potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data for genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification