Rhamnolipids: fermentation products of glucose with Pseudomonas putida, Pseudomonadaceae

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Rhamnolipids: fermentation products of glucose with Pseudomonas bacteria

EC Number:

943-175-7

IUPAC Name:

Rhamnolipids: fermentation products of glucose with Pseudomonas bacteria

Test material form:

solid: particulate/powder

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.

Method

Species / strain

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).

Controls

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.