Saccharomyces cerevisiae, lysate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the three in vitro key studies (Orovecz, Klimisch 1, GLP, 2018, OECD TG 471; Chevallier, Klimisch 1, GLP, 2018, OECD 487 ; Sire, Klimisch 1, GLP, 2018, OECD TG 490), the test substance Saccharomyces Cerevisiae, lysate did not induce mutagenicity and clastogenicity effects on mammalian cells or bacteria strain. Hence, according to CLP criteria, the test substance Saccharomyces Cerevisiae, lysate was not considered as mutagenic substance in vitro.

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:

From 6 October 2017 To 25 April 2018

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:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the sponsor, batch no. AC17F00560
- Expiration date of the lot/batch: February 2019
- Purity test date: 30 June 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from the light
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: precipitates were in solution at higher than or equal to 125 µg/mL
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Since the stock formulation used in the first experiment was heated at 60°C for 10 minutes whereas heating was not recommended, the corresponding results were not retained for the interpretation and are presented for information purpose only. A second cytogenetic experiment was then undertaken using an unheated formulation.
- Final dilution of a dissolved solid, stock liquid or gel: The test item was suspended in the vehicle at the concentrations of: 10 mg/mL for the preliminary cytotoxicity test, 5 mg/mL for the cytogenetic experiments.
- Final preparation of a solid: not applicable

FORM AS APPLIED IN THE TEST (if different from that of starting material)
In solution with water

Target gene:

micronuclei

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ATCC by the intermediate of Biovalley
- Suitability of cells: recommanded by international regulations
- Cell cycle length, doubling time or proliferation index: average cell cycle time : 10-12 hours
- Sex, age and number of blood donors if applicable: not applicable
- Whether whole blood or separated lymphocytes were used if applicable: not applicable
- Number of passages if applicable: not applicable
- Methods for maintenance in cell culture if applicable: Cell cultures were grown at 37°C in a humidified atmosphere of 5% CO2/95% air in culture medium. The culture medium was RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL),streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL). This medium was supplemented by heat-inactivated horse serum at 10% (v/v).
- Modal number of chromosomes: 40
- Normal (negative control) cell cycle time: 10-12 hours

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640, humidified atmosphere, at 37°C, 5%CO2/95% air
- Properly maintained: not specified
- Periodically checked for Mycoplasma contamination: not specified
- Periodically checked for karyotype stability: not specified
- Periodically 'cleansed' against high spontaneous background: not specified

Additional strain / cell type characteristics:

other: TK+/-

Metabolic activation:

with and without

Metabolic activation system:

The S9 mix consists of induced enzymatic systems contained in rat liver post-mitochondrial fraction (S9 fraction) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by intraperitoneal route.

Test concentrations with justification for top dose:

Based on available solubility data, the highest achievable dose level to be used in the preliminary cytotoxicity test was 500 µg/mL, dose level limited by the solubility of the test item in the vehicle and by the treatment volume. Thus, using a test item stock formulation at the concentration of 10 mg/mL in the vehicle (i.e. water for injections) and a maximum treatment volume of 5% (v/v) in the culture medium, the dose levels selected for the treatment of the preliminary test were: 1, 10, 50, 100, 250 and 500 µg/mL.

At the highest tested dose level of 500 µg/mL, the pH of the culture medium was approximately 7.4 and the osmolality was 291 mOsm/kg H2O (both as the vehicle control). Therefore, none of the selected dose levels was considered to produce extreme culture conditions and the dose level of 500 µg/mL could be selected as the highest dose level for the main experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:water
- Justification for choice of solvent/vehicle: According to available solubility data, the vehicle used for the preparation of test item dose formulations and the treatment of vehicle control cultures was water for injections.

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: colchicine, without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 3x10E5 cells/mL

DURATION
- Preincubation period: not applicable
- Exposure duration: 3 hours (with and without S9) and 24 hours (without S9)
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours after treatment or 27 hours after treatment (for 3 hours treatment period)

SELECTION AGENT (mutation assays): not applicable

SPINDLE INHIBITOR (cytogenetic assays): not used

STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: duplicates were used for each condition

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
After the final cell counting, the 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.

Depending on the observation at the end of the recovery period (presence or absence of precipitate and/or cytotoxicity), three dose levels of the test item-treated cultures were selected for spreading on slides. Cells were dropped onto clean glass slides. The slides were air-dried before being stained for approximately 15 min in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above.

NUMBER OF CELLS EVALUATED: 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose).

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Analysis was performed under a microscope (1000 x magnification), on the basis of the recommendations of Miller et al. (1995) (e), according to the following criteria:
-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.

Number of cells with micronuclei and number of micronuclei per cell were given separately for each treated and control culture.


DETERMINATION OF CYTOTOXICITY
- Method: Population Doubling
- Any supplementary information relevant to cytotoxicity: For each culture, the Population Doubling (PD) was calculated and used relative to that of the vehicle control. The population doubling is the log of the ratio of the final count at the time of harvesting (N) to the starting count (N0), divided by the log of 2.
PD = [log (N/N0)]/log 2.
Mean PD (%) = (Mean PD treated / Mean PD vehicle control) x 100
The cytotoxicity induced by a treatment was evaluated by the decrease in the PD, when compared to the vehicle control (Mean % PD of the vehicle control set to 100%).

Decrease in PD (%) = 100 - Mean PD as % of control

OTHER EXAMINATIONS:
- Determination of polyploidy: notspecified
- Determination of endoreplication: not specified
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): not specified

Evaluation criteria:

Acceptance criteria
Each main experiment was considered valid if the following criteria were met:
-the mean PD of the vehicle control had to be ≥ 1 (indicating that cells have undergone mitosis),
- the mean frequency of micronucleated cells in the vehicle control should be consistent with (but not necessary within) control historical data of the Laboratory. In any case, this frequency should be ≤ 5‰,
-a statistically significant increase in the frequency of micronucleated cells had to be obtained in the positive controls over the background frequency of the vehicle control cultures.

Statistics:

For each condition of the cytogenetic experiment, the frequency of micronucleated cells in treated cultures was compared to that of the vehicle control cultures.
This comparison was performed using the 2 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. This statistical analysis was performed using a validated Excel sheet.

To assess the dose-response trend, a linear regression was performed between the frequencies of micronucleated cells and the dose levels. This statistical analysis was performed using SAS Enterprise Guide software.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
At the highest tested dose level of 500 µg/mL, the pH of the culture medium was approximately 7.4 and the osmolality was 291 mOsm/kg H2O (both as the vehicle control). Therefore, none of the selected dose levels was considered to produce extreme culture conditions and the dose level of 500 µg/mL could be selected as the highest dose level for the main experiment.

At the end of both 3- and 24-hour treatment periods, a precipitate was observed in the culture medium at dose levels higher than or equal to 100 µg/mL.

No noteworthy cytotoxicity was observed at any dose levels, in any conditions, as shown by the absence of notable decrease in the PD relative to the corresponding vehicle control.

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: treated 83 to 160 x10E4 cells/mL ; control : 67 to 130 x10E4 cells/mL
- Indication whether binucleate or mononucleate where appropriate: not specified

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
See tables in any other information on results incl. tables section

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used:Population Doubling

Table 1. Second experiment without S9 mix, 3-h treatment + 24-h recovery: cytotoxicity

 

Treatment		Cell concentration used for treatment ( x 10^4cells/mL)	Culture	Post-treatment cell count (x 10^4cells/mL)	Mean PD	Mean PD as % of control	Decrease in (%)	PD
Vehicle control		30	C1 C2	92.0 100.0	1.7	100
Test item (µg/mL)
7.81		30	C1 C2	87.0 100.0	1.6	98	2
15.6		30	C1 C2	100.0 102.5	1.8	105	none
31.3		30	C1 C2	83.5 105.5	1.7	99	1
62.5		30	C1 C2	100.0 84.0	1.6	96	4
125	P	30	C1 C2	113.0 110.0	1.9	113	none
250	P	30	C1 C2	105.0 102.0	1.8	106	none
Positive controls
MMC ( 1 µg/mL )		30	C1 C2	38.0 38.3	0.3	21	79
COL ( 0.5 µg/mL)		30	C1 C2	34.0 24.2	#	#	#

PD: population doubling

Vehicle control: water for injections MMC:MitomycinC

COL:ColchicineC1:Culture1

C2:Culture2

#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment P: precipitate was noted in the culture medium at the end of treatment

 

Table 2 Second experiment without S9 mix, 3-h treatment + 24-h recovery: cytogenetic results

 

Treatment		Mean PD as % of control	Culture	Number of cells analysed	Number of cells with n micronuclei					Total micronucleatedcells		Frequency of micronucleatedcells (^0/00)	Ratio treated/control
					n = 1	n = 2	n = 3	n = 4	n = 5
										per culture	per dose
Vehicle control		100	C1 C2	1000 1000	1 1	0 0	0 0	0 0	0 0	1 1	2	1
Test item (µg/mL)
7.81		98	C1 C2
15.6		105	C1 C2
31.3		99	C1 C2	1000 1000	3 0	0 0	0 0	0 0	0 0	3 0	3	2	1.5
62.5		96	C1 C2	1000 1000	2 1	0 0	0 0	0 0	0 0	2 1	3	2	1.5
125	P	113	C1 C2	1000 1000	3 0	0 0	0 0	0 0	0 0	3 0	3	2	1.5
250	P	106	C1 C2
Positive controls
MMC ( 1 µg/mL )		21	C1 C2	1000 1000	52 37	13 7	5 0	0 2	0 0	70 46	116	58	58.0	***
COL ( 0.5 µg/mL)		#	C1 C2	1000 1000	14 16	1 1	0 0	0 0	0 0	15 17	32	16	16.0	***

PD:populationdoubling                                                                                                                                                                                                             Statistics:2 x 2 contingencytable:

Vehicle control: waterforinjections                                                                                                                                                                                          ***: p <0.001

MMC:MitomycinCCOL:ColchicineC1:Culture1

C2:Culture2

#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment P: precipitate was noted in the culture medium at the end of treatment

 

Table 3. Second experiment without S9 mix, 24-h treatment + 0-h recovery: cytotoxicity

 

Treatment		Cell concentration used for treatment ( x 10^4cells/mL)	Culture	Post-treatment cell count (x 10^4cells/mL)	Mean PD	Mean PD as % of control	Decrease in (%)	PD
Vehicle control		30	C1 C2	130.0 113.0	2.0	100
Test item (µg/mL)
7.81		30	C1 C2	136.0 119.0	2.1	103	none
15.6		30	C1 C2	138.0 128.0	2.1	106	none
31.3		30	C1 C2	160.0 134.0	2.3	114	none
62.5		30	C1 C2	120.0 135.0	2.1	103	none
125	P	30	C1 C2	136.0 132.0	2.2	107	none
250	P	30	C1 C2	133.0 133.0	2.1	106	none
Positive controls
MMC (1 µg/mL )		30	C1 C2	50.7 45.0	0.7	33	67
COL (0.5 µg/mL)		30	C1 C2	28.8 29.5	#	#	#

PD: population doubling

Vehicle control: water for injections MMC:MitomycinC

COL:ColchicineC1:Culture1

C2:Culture2

#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment P: precipitate was noted in the culture medium at the end of treatment

 

Table 4. Second experiment without S9 mix, 24-h treatment + 0-h recovery: cytogenetic results

 

Treatment		Mean PD as % of control	Culture	Number of cellsanalysed	Number of cells with n micronuclei					Total micronucleatedcells		Frequency of micronucleatedcells (^0/00)	Ratio treated/control
					n = 1	n = 2	n = 3	n = 4	n = 5
										per culture	per dose
Vehicle control		100	C1 C2	1000 1000	0 0	0 0	0 0	0 0	0 0	0 0	0	0	a)
Test item (µg/mL)
7.81		103	C1 C2
15.6		106	C1 C2
31.3		114	C1 C2	1000 1000	1 1	0 0	0 0	0 0	0 0	1 1	2	1	2.0
62.5		103	C1 C2	1000 1000	2 0	0 0	0 0	0 0	1 0	3 0	3	2	3.0
125	P	107	C1 C2	1000 1000	3 1	0 0	0 0	0 0	0 0	3 1	4	2	4.0
250	P	106	C1 C2
Positive controls
MMC (1 µg/mL )		33	C1 C2	1000 1000	12 23	1 3	0 0	0 0	0 0	13 26	39	20	39.0	***
COL (0.5 µg/mL)		#	C1 C2	1000 1000	8 3	2 0	0 0	0 0	0 0	10 3	13	7	13.0	***

PD:populationdoubling                                                                                                                                                                                                             Statistics:2 x 2 contingencytable:

Vehicle control: waterforinjections                                                                                                                                                                                          ***: p <0.001

MMC:MitomycinCCOL:ColchicineC1:Culture1

C2:Culture2

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

a)therawdata obtainedforthevehiclecontrol is 0 but itwaschanged to 1 inorderto allow calculation of ratios P: precipitatewasnoted in the culture medium at the end oftreatment

 

Table 5. Second experiment with S9 mix, 3-h treatment + 24-h recovery: cytotoxicity

 

Treatment		Cell concentration used for treatment ( x 10^4cells/mL)	Culture	Post-treatment cell count (x 10^4cells/mL)	Mean PD	Mean PD as % of control	Decrease in (%)	PD
Vehicle control		30	C1 C2	116.0 100.0	1.8	100
Test item (µg/mL)
7.81		30	C1 C2	110.0 111.0	1.9	102	none
15.6		30	C1 C2	123.0 109.0	2.0	106	none
31.3		30	C1 C2	129.0 122.0	2.1	112	none
62.5		30	C1 C2	120.0 100.0	1.9	101	none
125	P	30	C1 C2	113.0 111.0	1.9	103	none
250	P	30	C1 C2	106.0 102.0	1.8	97	3
Positive controls
CPA (6 µg/mL )		30	C1 C2	69.5 64.0	1.2	62	38

PD: population doubling

Vehicle control: water for injections CPA: Cyclophosphamide

C1:Culture1

C2:Culture2

P: precipitate was noted in the culture medium at the end of treatment

 

Table 6. Second experiment with S9 mix, 3-h treatment + 24-h recovery: cytogenetic results

 

Treatment		Mean PD as % of control	Culture	Number of cells analysed	Number of cells with n micronuclei					Totalmicronucleatedcells		Frequency ofmicronucleatedcells (^0/00)	Ratio treated/control
					n = 1	n = 2	n = 3	n = 4	n = 5
										per culture	per dose
Vehicle control		100	C1 C2	1000 1000	1 1	0 0	0 0	0 0	0 0	1 1	2	1
Test item (µg/mL)
7.81		102	C1 C2
15.6		106	C1 C2
31.3		112	C1 C2	1000 1000	2 3	0 0	0 0	0 0	0 0	2 3	5	3	2.5
62.5		101	C1 C2	1000 1000	2 1	0 0	0 0	0 0	0 0	2 1	3	2	1.5
125	P	103	C1 C2	1000 1000	2 0	0 0	0 0	0 0	0 0	2 0	2	1	1.0
250	P	97	C1 C2
Positive controls
CPA (6 µg/mL )		62	C1 C2	1000 1000	41 33	1 2	0 0	0 0	0 0	42 35	77	39	38.5	***

PD:populationdoubling                                                                                                                                                                                                             Statistics:2 x 2 contingencytable:

Vehicle control: waterforinjections                                                                                                                                                                                          ***: p <0.001

CPA:CyclophosphamideC1:Culture1

C2:Culture2

P: precipitate was noted in the culture medium at the end of treatment

 

Table 7: Historical data of experiments without S9

Parameter	Frequency ofmicronucleatedcells in 1000 cells
Treatmentcontrol	3hourstreatment+ 24hoursrecovery			24hourstreatment+ 0hoursrecovery
Control items	Vehicle(control)	MMC (1µg/mL)	COL (0.5 µg/mL)	Vehicle(control)	MMC (1µg/mL)	COL (0.5 µg/mL)
n	50	50	50	28	28	28
mean	1.7	133	22.9	2.1	46.4	34.5
SD	1	67.5	13.3	1.1	19.8	17.5
LowerCL95%	1.4	113.8	19.1	1.6	38.7	27.7
UpperCL 95%	1.9	152.2	26.6	2.5	54.1	41.2
5th percentile	0.5	23	7.5	0	11.5	14.5
Median	1.5	138.3	20	2	49.5	27.0
95th percentile	4	238.5	54.5	3.5	72.5	65.5
Min	0	18	5.5	0	8	10.0
Max	4.5	306	65	5	97.5	75.0

 

Table 8: Historical data of experiments with S9

Parameter	Frequency of micronucleated cells in 1000 cells
Treatmentcontrol	3hourstreatment+ 24hoursrecovery
Control items	Vehiclecontrol	CPA (6µg/mL)
n	67	67
mean	1.5	101.9
SD	0.9	49.3
LowerCL95%	1.3	89.8
UpperCL 95%	1.7	113.9
5th percentile	0.5	26
Median	1.5	105.5
95th percentile	3.0	184.5
Min	0	14
Max	3.5	251

 

 

Conclusions:

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

Executive summary:

The objective of this GLP-compliant study was to evaluate the potential of the test item, Saccharomyces cerevisiae, lysate, to induce an increase in the frequency of micronucleated cells in the mouse cell line L5178Y TK+/- according to OECD TG 487 method.

After a preliminary cytotoxicity test, the potential of the test item Saccharomyces cerevisiae, lysate, suspended in water for injections, to induce an increase in the frequency of micronucleated cells was evaluated in a main cytogenetic experiment, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254 as follows:

-without S9 mix, 3 hours treatment and 24 hours recovery

- without S9 mix, 24 hours treatment, no recovery

-with S9 mix, 3 hours treatment, 24 hours recovery

Each treatment was coupled to an assessment of cytotoxicity at the same dose levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells.

After the final cell counting, the cells were washed and fixed. Then, cells from three dose levels of the test item-treated cultures were dropped onto clean glass slides. The slides were air-dried before being stained in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared. For each main experiment (with or without S9 mix), micronuclei were analyzed  for  three  dose  levels   of   the  test item,  for  the  vehicle  and  the  positive  controls,   in   1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose). Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.

Since the test item was found poorly soluble in the culture medium and  non-cytotoxic in the preliminary test, the selection of the highest dose level for the main experiment was based on the presence of precipitate, according to the criteria specified in the international regulations.The mean population doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells.

With a treatment volume of 5 % (v/v) in culture medium, the dose levels selected for the treatment were: 7.81, 15.6, 31.3, 62.5, 125 and 250 µg/mL for the 3-hour treatments with and without S9 mix, as well as for the 24-hour treatment without S9 mix. At the end of both 3- and 24-hour treatment periods, a precipitate was observed in the culture medium at dose levels >= 125 µg/mL. No noteworthy cytotoxicity was induced at any dose levels, either following the 3-hour treatments with and without S9 mix or the 24-hour treatment without S9 mix, as shown by the absence of notable decrease in the PD relative to the corresponding vehicle control.

For the three experimental conditions, the dose levels selected for the micronucleus analysis were: 31.3, 62.5 and 125 µg/mL, the latter being the lowest dose level showing precipitate in the culture medium at the end of the treatment periods. Following the 3-hour treatments with and without S9 mix or the 24-hour treatment without S9 mix, neither statistically significant nor dose-related increase in the frequency of micronucleated cells was noted at any of the analyzed dose levels relative to the corresponding vehicle control.

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

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 14 August 2017 to 1st February

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (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:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AC17F00560
- Expiration date of the lot/batch: February 2019
- Purity test date: 30 June 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25°CC, ≤70 RH%**)
- Solubility and stability of the test substance in the solvent/vehicle: the test item was soluble in distilled water (see attached table "The solubility of the test item in distilled water" in "Any information on material and method including table" section )
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The appropriate vehicle (solvent) and the behaviour of the test item formulations with the solution of top agar and phosphate buffer were examined in a preliminary compatibility test. Distilled water was used as solvent to prepare the stock solution of the test material. Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution (50 mg/mL) using the selected solvent and were used within 4 hours after preparation.
- Final dilution of a dissolved solid, stock liquid or gel: 50, 15.81, 5, 1.581, 0.5, 0.1581, 0.05 mg/mL respectively for 5000, 1581, 500, 158.1, 50, 15.81 and 5 µg/plate
- Final preparation of a solid: not applicable

FORM AS APPLIED IN THE TEST (if different from that of starting material) diluted in distilled water

Target gene:

histidine locus

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):

Not applicable

Additional strain / cell type characteristics:

other: See table 2 "Genotypes of the strains used for mutagenicity testing" in "additional information on material and methods includ. tables" section

Metabolic activation:

with and without

Metabolic activation system:

S9 mitochrondrial section from Wistar rats liver induced with phenobarbital and Beta-Naphthoflavone at 80mg/kg/day by oral gavage during 3 days

Test concentrations with justification for top dose:

Based on the results of the preliminary tests, a 50 mg/mL stock solution was prepared in Distilled water. Maximum seven test concentrations were prepared by successive dilutions of the stock solution, to obtain lower doses. The maximum test concentration was 5000 μg test item/plate. Examined concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate.
Examined concentrations in the Confirmatory Mutation Test were 5000, 1581, 500,158.1, 50, 15.81 and 5 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: The solubility of the test item was examined using Distilled water, N,NDimethylformamide (DMF), Acetone and Dimethyl sulfoxide (DMSO). At the 100 mg/mL concentration, suspension with quick sedimentation was observed using DMSO, DMF and Acetone. At this concentration, suspension with slower sedimentation was observed using Distilled water. At the 50 mg/mL concentration, yellow homogeneous suspension with slower sedimentation was observed using Distilled water.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

True negative controls:

yes

Remarks:

Untreated

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 2-Aminoanthracene ; 4-nitro-1,2-phenylenediamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (initial); preincubation (confirmatory)
- Cell density at seeding (if applicable): not applicable

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours

NUMBER OF REPLICATIONS: triplicates

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: not applicable The colony numbers on the untreated / negative (solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.

NUMBER OF CELLS EVALUATED:
not applicable

DETERMINATION OF CYTOTOXICITY
Cytotoxicity was evaluated in initial mutation test by inhibition of background lawn of auxotrophic cells method.

Evaluation criteria:

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

Statistics:

statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

Key result

Species / strain:

S. typhimurium TA 1535

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

Key result

Species / strain:

S. typhimurium TA 1537

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

Key result

Species / strain:

S. typhimurium TA 98

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

Key result

Species / strain:

S. typhimurium TA 100

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

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

not valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Based on the solubility test, a 50 mg/mL stock solution was prepared in Distilled water. Seven test concentrations were prepared by successive dilutions of the stock solution, spaced by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98 and TA100) were determined at concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item, in the absence and presence of metabolic activation.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: inhibition of background lawn of auxotrophic cells: No cytotoxicity was observed.

Table 3:Summary Table of the Initial Mutation Test

 

Concentrations (mg/plate)	Mean values ofrevertants/ Mutation factor (MF)	Salmonella typhimuriumtester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	17.3	22.0	71.3	73.0	10.3	13.7	12.0	12.3	45.0	51.7
	MF	1.02	0.94	0.96	1.03	0.91	1.28	0.88	1.16	1.01	0.97
DMSO control	Mean	15.7	21.7	--	76.3	--	12.7	13.0	10.0	--	46.7
	MF	0.92	0.93	--	1.08	--	1.19	0.95	0.94	--	0.88
Distilled water control	Mean	--	--	73.3	--	11.7	--	--	--	47.7	--
	MF	--	--	0.99	--	1.03	--	--	--	1.07	--
Distilled water control 100µl	Mean	17.0	23.3	74.0	71.0	11.3	10.7	13.7	10.7	44.7	53.3
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	13.3	22.0	62.7	78.0	12.7	11.3	13.3	11.7	64.7	69.0
	MF	0.78	0.94	0.85	1.10	1.12	1.06	0.98	1.09	1.45	1.29
1581	Mean	21.0	22.7	68.3	82.3	12.3	12.0	12.0	14.0	65.3	66.3
	MF	1.24	0.97	0.92	1.16	1.09	1.13	0.88	1.31	1.46	1.24
500	Mean	15.3	20.3	69.0	78.7	13.0	11.7	12.0	12.0	63.0	64.7
	MF	0.90	0.87	0.93	1.11	1.15	1.09	0.88	1.13	1.41	1.21
158.1	Mean	16.3	22.0	73.0	80.7	14.7	11.7	10.7	10.3	64.7	66.3
	MF	0.96	0.94	0.99	1.14	1.29	1.09	0.78	0.97	1.45	1.24
50	Mean	18.3	21.3	79.3	84.7	14.0	9.3	11.3	15.0	58.0	67.3
	MF	1.08	0.91	1.07	1.19	1.24	0.88	0.83	1.41	1.30	1.26
15.81	Mean	16.0	25.7	81.7	81.7	13.3	11.0	10.7	12.0	57.7	66.3
	MF	0.94	1.10	1.10	1.15	1.18	1.03	0.78	1.13	1.29	1.24
NPD (4mg)	Mean	404.0	--	--	--	--	--	--	--	--	--
	MF	25.79	--	--	--	--	--	--	--	--	--
2AA (2mg)	Mean	--	2474.7	--	2418.7	--	206.3	-	215.3	-	-
	MF	--	114.22	--	31.69	--	16.29	-	21.53	-	-
2AA (50mg)	Mean	--		--	--	--	--	--	--	--	257.3
	MF	--	--	--	--	--	--	--	--	--	5.51
SAZ (2mg)	Mean	--	--	1142.0	--	1026.7	--	--	--	--	--
	MF	--	--	15.57	-	88.00	--	--	--	--	--
9AA (50mg)	Mean	--	--	--	--	--	--	421.3	--	--	--
	MF	--	--	--	--	--	--	32.41	--	--	--
MMS (2mL)	Mean	--	--	--	--	--	--	--	--	1008.7	--
	MF	--	--	--	--	--	--	--	--	21.16	--

Table 4:Summary Table of the Confirmatory Mutation Test

 

Concentrations (mg/plate)	Mean valuesofrevertants/ Mutation factor(MF)	Salmonella typhimuriumtester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	22.3	27.0	101.0	109.3	10.3	12.3	13.7	14.0	53.0	53.0
	MF	1.02	1.03	1.05	1.05	0.74	0.93	0.95	1.02	0.93	1.02
DMSO control	Mean	21.3	25.7	--	99.3	--	13.7	11.3	12.3	--	56.0
	MF	0.97	0.97	--	0.96	--	1.03	0.79	0.90	--	1.08
Distilled water control	Mean	--	--	102.3	--	13.0	--	--	--	53.0	--
	MF	--	--	1.06	--	0.93	--	--	--	0.93	--
Distilled water control 100µl	Mean	22.0	26.3	96.3	103.7	14.0	13.3	14.3	13.7	57.0	52.0
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	23.0	24.7	83.3	109.3	14.3	14.0	16.0	12.7	43.3	52.7
	MF	1.05	0.94	0.87	1.05	1.02	1.05	1.12	0.93	0.76	1.01
1581	Mean	22.7	21.0	95.3	122.7	14.0	13.7	14.3	18.0	42.7	50.3
	MF	1.03	0.80	0.99	1.18	1.00	1.03	1.00	1.32	0.75	0.97
500	Mean	23.3	28.3	110.3	109.0	15.3	15.0	15.0	13.3	45.7	48.0
	MF	1.06	1.08	1.15	1.05	1.10	1.13	1.05	0.98	0.80	0.92
158.1	Mean	19.0	22.7	107.7	111.7	14.7	14.3	13.0	14.3	41.7	50.7
	MF	0.86	0.86	1.12	1.08	1.05	1.08	0.91	1.05	0.73	0.97
50	Mean	22.3	25.0	109.3	112.0	15.3	13.3	12.7	14.3	47.0	51.0
	MF	1.02	0.95	1.13	1.08	1.10	1.00	0.88	1.05	0.82	0.98
15.81	Mean	23.3	26.7	110.3	110.3	9.0	13.3	14.7	14.0	43.0	46.7
	MF	1.06	1.01	1.15	1.06	0.64	1.00	1.02	1.02	0.75	0.90
5	Mean	17.7	27.3	102.3	116.0	15.0	14.7	13.7	17.0	44.3	47.0
	MF	0.80	1.04	1.06	1.12	1.07	1.10	0.95	1.24	0.78	0.90
NPD (4mg)	Mean	394.7	--	--	--	--	--	--	--	--	--
	MF	18.50	--	--	--	--	--	--	--	--	--
2AA (2mg)	Mean	--	2402.7	--	2421.3	--	209.7	--	204.0	--	--
	MF	--	93.61	--	24.38	--	15.34	--	16.54	--	--
2AA (50mg)	Mean	--	--	--	--	--	--	--	--	--	265.0
	MF	--	--	--	--	--	--	--	--	--	4.73
SAZ (2mg)	Mean	--	--	1177.3	--	1054.7	--	--	--	--	--
	MF	--	--	11.50	--	81.13	--	--	--	--	--
9AA (50mg)	Mean	--	--	--	--	--	--	425.3	--	--	--
	MF	--	--	--	--	--	--	37.53	--	--	--
MMS (2mL)	Mean	--	--	--	--	--	--	--	--	1048.0	--
	MF	--	--	--	--	--	--	--	--	19.77	--

 

Table 5 Historical Control Data (Period of 2011-2016)

Untreated control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	22.7	103.6	11.8	7.2	33.4	29.7	111.7	11.5	8.9	39.1
St. dev.	5.8	21.4	5.1	3.3	9.7	6.8	19.6	3.9	3.8	9.9
Range	9-50	54-210	1-46	1-24	11-82	10-56	65-204	1-39	1-29	16-89
n	1371	1357	1365	1371	1374	1377	1365	1373	1380	1371
DMSO control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	21.7	98.9	12.0	7.1	32.3	28.7	109.5	11.3	8.7	38.1
St. dev.	5.7	20.7	5.0	3.3	9.6	7.0	20.7	3.8	3.7	9.7
Range	6-55	40-217	1-43	1-25	7-81	11-67	53-229	2-33	1-29	9-85
n	1482	1473	1479	1485	1482	1487	1476	1487	1491	1482
Distilled water control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	23.5	103.2	11.9	7.8	34.5	30.8	112.2	11.3	9.3	40.3
St. dev.	6.0	22.4	4.9	3.4	9.8	7.1	21.8	3.7	3.7	10.0
Range	11-45	45-215	2-47	2-24	12-84	10-53	64-222	3-39	1-24	13-91
n	267	1359	1365	270	1392	267	1371	1380	267	1383
DMF control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	20.4	89.9	11.2	6.9	34.7	28.1	100.3	11.0	8.0	38.0
St. dev.	5.6	17.8	4.7	3.1	12.3	7.0	19.2	3.6	3.1	10.2
Range	8-38	54-152	1-34	1-19	16-99	13-49	60-156	3-21	1-23	17-76
n	216	216	216	216	207	216	216	216	213	207
Acetone control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	22.6	98.1	12.1	7.4	35.0	29.1	108.1	11.1	8.6	40.5
St. dev.	5.1	15.4	5.8	2.9	9.3	6.7	14.2	3.4	3.3	9.0
Range	11-39	62-160	4-49	1-17	17-62	15-52	66-177	4-22	1-19	17-69
n	278	279	279	279	276	279	279	282	279	279
Positive reference control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	357.2	1229.3	1169.8	454.1	1034.3	2410.2	2429.6	235.1	221.3	257.4
St. dev.	113.8	207.5	204.2	169.7	141.7	317.7	291.6	135.9	56.2	113.4
Range	152-2336	536-2120	208-2440	149-2104	488-1708	312-4918	1192-5240	101-2216	117-838	125-2512
n	1371	1359	1365	1371	1377	1378	1365	1377	1380	1371

TA98:SalmonellatyphimuriumTA98, TA100:SalmonellatyphimuriumTA100, TA1535:SalmonellatyphimuriumTA1535, TA1537:

Salmonella typhimuriumTA1537, E. coli:Escherichia coliWP2uvrA;n: number of cases

 

 

Conclusions:

Under the experimental condition of the study, the registered substance saccharomyces cerevisiae, lysate did not induced reverse mutation when incubated with bacteria strain. Hence, the registered substance was considered to be not mutagenic for bacteria strain according to CLP criteria.

Executive summary:

The purpose of this GLP compliant study was to evaluate the mutagenic potential of the test item by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli WP2 uvrA strain in the presence and absence of activated rat liver S9 fraction (performed according to OECD TG 471 method).

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using  histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/-naphthoflavone- induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).

Based on the results of the Compatibility Test, the test  item  was  dissolved  in  Distilled water at a concentration of 50 mg/mL. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 µg/plate were examined in the Range Finding Test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test item concentrations  in  the  Initial  Mutation  Test  were  5000,  1581,  500,  158.1,  50  and 15.81 μg/plate,  in  the  Confirmatory Mutation Test  were 5000, 1581, 500, 158.1, 50,

15.81 and 5 μg/plate.

Precipitate/slight precipitate was detected on the plates in the Preliminary experiment at Salmonella typhimurium TA98 strain and in the Confirmatory Mutation Test in all examined strains with and without metabolic activation at higher concentrations.

Inhibitory, cytotoxic effect of the test item was not detected in the Initial Mutation Test and Confirmatory Mutation Test.

In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

In conclusion, the test item Saccharomyces cerevisiae, lysate has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2 November 2017 to 14 December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

adopted on 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
supplied by the sponsor, batch no. AC17F00560
- Expiration date of the lot/batch: February 2019
- Purity test date: 30 June 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protected from humidity (kept in its original packaging)
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: precipitate was observed in the culture medium at dose levels >= 55.6 µg/mL
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was suspended in the vehicle.
- Final dilution of a dissolved solid, stock liquid or gel: -10 mg/mL for the preliminary cytotoxicity test; -2 mg/mL for the first mutagenicity experiment.
- Final preparation of a solid: The stock formulation was sonicated for 10 minutes and then magnetically stirred for at least 15 minutes at room temperature before use. The stock formulations and their dilutions were prepared within one hour before use, and then kept under magnetic stirring, at room temperature, until the end of use.

FORM AS APPLIED IN THE TEST (if different from that of starting material) in solution with water

Target gene:

Thymidine Kinase Locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ATCC (USA) through Biovalley (France)
- Suitability of cells: cell line recommended by international regulations for use in the in vitro mammalian cell gene mutation test
- Cell cycle length, doubling time or proliferation index: average cell cycle time : 10-12 hours
- Sex, age and number of blood donors if applicable: not applicable
- Whether whole blood or separated lymphocytes were used if applicable: not applicable
- Number of passages if applicable: not applicable
- Methods for maintenance in cell culture if applicable: The cells were stored in a cryoprotective medium [10% horse serum and 10% dimethylsulfoxide (DMSO)] at -80°C. 5% CO2 humidified incubator. The cells were maintained in flasks as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 10% v/v, in a 37°C, 5% CO2 humidified incubator.
- Modal number of chromosomes: 40
- Normal (negative control) cell cycle time: average cell cylcle time 10-12 hours

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The cells were maintained in flasks as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 10% v/v, in a 37°C, 5% CO2 humidified incubator.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability:not specified
- Periodically 'cleansed' against high spontaneous background:not specified

Additional strain / cell type characteristics:

other: TK +/-

Metabolic activation:

with and without

Metabolic activation system:

The metabolic activation system used in this study, S9 mix, consists of induced enzymatic systems contained in rat liver microsomal fraction (S9 fraction), obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route

Test concentrations with justification for top dose:

Preliminary cytotoxicity test : Based on available solubility data, the highest selected dose level was 500 µg/mL since it was expected to produce precipitation in the culture medium. This dose level was obtained using a test item concentration of 10 mg/mL and a treatment volume of 5% (v/v) in the culture medium (i.e. 1000 µL/20 mL culture medium for the 3-hour treatments and 2500 µL/50 mL culture medium for the 24 hour treatment).
The selected dose levels were 2.06, 6.17, 18.5, 55.6, 166.7 and 500 µg/mL.
Main experiments : Since the test item was found non-cytotoxic but poorly soluble in the culture medium, the highest dose level selection for the main experiment was based on the level of precipitate, according to the criteria specified in the international guidelines.
The selected dose levels were 3.13, 6.25, 12.5, 25, 50 and 100 µg/mL for each experiment with or without S9 mix.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: according to available solubility data

Untreated negative controls:

yes

Remarks:

vahicle control : water

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 2x 10E5 cells/mL (for mutagenicity experiment) and were seed in order to average of 1.6 cells per well

DURATION
- Preincubation period: cells were cutured for 2 day before mutagenicity experiment
- Exposure duration: 3 hours and 24 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 days and 3 hours (3 hours exposure period) or 15 days (24 hours exposure period)

SELECTION AGENT (mutation assays): Trifluorothymidine

NUMBER OF REPLICATIONS: duplicates were used per condition

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: not applicable

NUMBER OF CELLS EVALUATED: mutant colony were counted

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): not applicable

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Any supplementary information relevant to cytotoxicity:
For the evaluation of cytotoxicity, the following parameters were determined: Daily cell growth on Day 1 (or 2)= cell concentration on Day 1 (or 2)/ adjusted concentration on Day 0 (or 1)  2 x 10E5
Cell growth during the 24-hour treatment = post-treatment cell concentration / seeded cell concentration (i.e. = 2 x 10E5 cells/mL)
Suspension growth (SG) for 3-hour treatment = daily growth on Day 1 x daily growth on Day 2
Suspension growth (SG) for 24-hour treatment =cell growth during the 24-hour treatment x daily growth on Day 1 x daily growth on Day 2
RSG (relative SG) = SG treated / SG vehicle control x 100
Adjusted RSG= cell count factor x [(SG treated / SG vehicle control) x 100]
Cell count factor = treated post-treatment cell count/ vehicle control post-treatment cell count
Adjusted relative total growth = Adj. RSG x RCE2 / 100
RCE2 = cloning efficiency treated / cloning efficiency vehicle control x 100

The relative Mutant Frequency (MF) and the induced mutation frequency (IMF) were calculated as follows:

MF = CEmutant x 10E6 / CE2

IMF = MF treated – MF vehicle control

OTHER EXAMINATIONS:
- the clones were counted using a magnifying glass, differentiating small and large colonies:
-size of small colonies: < 25% of the diameter of the well,
-size of large colonies: > 25% of the diameter of the well.

Evaluation criteria:

IWGT recommendations were followed for the determination of a positive result, which should fulfill the following criteria:
- at least at one dose level the mutation frequency minus the mutation frequency of the vehicle control (IMF) equals or exceeds the global evaluation factor (GEF) of 126 x 10-6,
- a dose-response relationship is demonstrated by a statistically significant trend test.

Unless an effect is considered as clearly positive, the reproducibility of a positive effect should be confirmed.

Noteworthy increases in the mutation frequency observed only at high levels of cytotoxicity (Adj. RTG lower than 10%), but with no evidence of mutagenicity at dose levels with Adj. RTG between 10 and 20%, are not considered as positive results.
A test item may be considered as non-mutagenic when there is no culture showing an Adj. RTG value between 10 and 20% if (g):
- there is at least one negative data point between 20 and 25% Adj. RTG and no evidence of mutagenicity in a series of data points between 100 and 20% Adj. RTG,
- there is no evidence of mutagenicity in a series of data points between 100 and 25% and there is also a negative data point between 10 and 1% Adj. RTG.

Statistics:

To assess the dose-response relationship, a linear regression was performed between dose levels and individual mutation frequencies obtained at dose levels showing a mean Adj. RTG ≥ 10%

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
At the end of the treatment periods (3- or 24-hour treatment), a precipitate was observed in the culture medium at dose levels ≥ 55.6 µg/mL.

No cytotoxicity was induced at any of the tested dose levels, either with or without S9 mix (3- or 24-hour treatment), as shown by the absence of noteworthy decrease in the Adj. RTG.

RESULTS OF THE MAIL TEST:
1) Without S9 mix
- Cytotoxicity: Following either the 3- or 24-hour treatment, no cytotoxicity was observed at any of the tested dose levels, as shown by the absence of any noteworthy decrease in the Adj. RTG.
- Mutagenicity: Following either the 3- or 24-hour treatment, no increase in the mutation frequency exceeding the GEF was observed at any of the tested dose levels relative to the corresponding vehicle control and no dose-response relationship was demonstrated by the linear regression. These results did not meet the criteria for a positive response (Tables 1 and 2 in "Any other information on results incl. Tables" section).

2) With S9 mix
1) Cytotoxicity: No cytotoxicity was observed at any of the tested dose levels, as shown by the absence of any noteworthy decrease in the Adj. RTG
2) Mutagenicity: Relative to the vehicle control, no increase in the mutation frequency exceeding the GEF was observed at any of the tested dose levels and no dose-response relationship was demonstrated by the linear regression. These results did not meet the criteria for a positive response (Table 3 in "Any other information on results incl. Tables" section).

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No effect of the pH: 7.4
- Effects of osmolality: No effect on osmolality, Osmolality : 290-299 mOsm/kg H2O
- Evaporation from medium: not applicable
- Water solubility: soluble until 50µg/mL (main terst)
- Precipitation: A precipitate was noted in the culture medium at the end of the treatment periods at dose levels ≥ 50 µg/mL (Main Test).


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: 3 hours treatment without S9 mix MMS : 652-1426 ; mean : 1043±216 ; Lower CL 95% : 935 - Upper CL 95% : 1150
3 hours treatment without S9 mix MMS :401-1013 ; mean : 649±174 ; Lower CL 95% : 563 - Upper CL 95% : 735
CPA with S9 mix 3 hours treatment: 479 - 2269 ; mean : 1113±441 ; Lower CL 95% :938 - Upper CL 95% : 1287
- Negative (solvent/vehicle) historical control data: 3 hours treatment without S9 mix : 71-152 ; mean 109 ±22 ; Lower CL 95% :98 - Upper CL 95% : 120
24 hours treatment without S9 mix : 73 - 143; mean 105 ± 23 ; Lower CL 95% : 94 - Upper CL 95% : 117
3 hours treatment with S9 mix : 59-174 ; mean 110 ±29 ; Lower CL 95% : 99 - Upper CL 95% : 122

Table1.Mutagenicity experiment without S9 mix, 3-hour treatment: mutagenicity results

Doses µg/mL		Adj. RTG %	CE2	Wells*					MFx10^-6	Ratio MF	Mean values of C1and C2
											Adj RTG%	CE2	MFx10^-6		IMF
0	C1	100	0.64	EW	79	78	82	83	138	1.0
				LC	9	8	10	7	72	1.0
				SC	8	10	5	6	61	1.0
	C2	100	0.76	EW	67	72	68	83	185	1.0	100	0.70		161
				LC	23	14	18	7	116	1.0			LC	92
				SC	7	10	11	6	61	1.0			SC	61
3.13	C1	127	0.68	EW	72	76	86	70	173	1.3
				LC	14	14	6	12	94	1.3
				SC	12	6	4	14	73	1.2
	C2	125	0.85	EW	67	71	77	83	149	0.8	126	0.77		161	none
				LC	17	17	12	9	91	0.8			LC	92	none
				SC	12	8	8	4	51	0.8			SC	62	1
6.25	C1	152	1.20	EW	66	80	67	75	120	0.9
				LC	16	10	20	13	69	1.0
				SC	16	7	10	9	48	0.8
	C2	117	0.76	EW	69	76	76	72	178	1.0	134	0.98		149	none
				LC	15	15	15	17	116	1.0			LC	93	none
				SC	12	5	5	7	52	0.8			SC	50	none
12.5	C1	179	1.16	EW	74	72	80	72	109	0.8
				LC	9	13	8	7	44	0.6
				SC	13	11	9	7	47	0.8
	C2	74	0.66	EW	73	67	87	84	160	0.9	126	0.91		135	none
				LC	16	20	6	7	104	0.9			LC	74	none
				SC	8	12	4	5	60	1.0			SC	54	none
25	C1	178	1.00	EW	78	75	69	81	119	0.9
				LC	15	14	15	6	70	1.0
				SC	4	8	13	9	47	0.8
	C2	114	0.82	EW	69	63	70	83	183	1.0	146	0.91		151	none
				LC	16	25	16	8	114	1.0			LC	74	none
				SC	12	9	10	5	60	1.0			SC	54	none
50	C1	161	0.91	EW	65	80	80	71	144	1.0
				LC	17	10	8	13	74	1.0
				SC	16	7	8	12	65	1.1
	C2	94	0.71	EW	74	13	72	75	351	1.9	127	0.81		247	86
				LC	12	75	11	13	241	2.1			LC	158	63
				SC	10	12	14	9	88	1.4			SC	77	16
100	C1	168	1.03	EW	73	74	66	77	136	1.0
				LC	13	13	17	10	72	1.0
				SC	11	10	13	9	58	0.9
	C2	112	0.91	EW	56	55	75	82	198	1.1	140	0.97		167	6
				LC	35	25	13	8	131	1.1			LC	101	7
				SC	6	16	8	6	54	0.9			SC	56	none
MMS 25 µg/mL	C1	85	0.57	EW	40	44	42	39	740	5.4
				LC	23	24	24	27	258	3.6
				SC	37	34	39	40	434	7.1
	C2	28	0.36	EW	44	42	43	48	1077	5.8	56	0.47		908	747
				LC	25	22	24	24	395	3.4			LC	327	232
				SC	31	36	30	24	526	8.6			SC	480	419

Adj. RTG: adjusted relative total growth                                         C1: culture 1

CE2: cloning efficiency                                                                  C2: culture 2

*: empty wells are counted on a total number of 96wells               0: vehicle control (water for injections)

MF: mutation frequency                                                                  MMS: methyl methane sulfonate

IMF: Induced Mutation Frequency                                                  

P: precipitate observed in the culture medium at the end of treatment

LC: wells with large colonies

SC: wells with small colonies

EW: empty wells

 

Table2.Mutagenicity experiment without S9 mix, 24-hour treatment: mutagenicity results

Doses µg/mL		Adj. RTG %	CE2	Wells*					MFx10^-6	Ratio MF	Mean values of C1and C2
											Adj RTG%	CE2	MFx10^-6		IMF
0	C1	100	0.96	EW	84	85	85	83	68	1.0
				LC	8	7	6	10	44	1.0
				SC	4	4	5	3	22	1.0
	C2	100	1.12	EW	88	84	85	81	57	1.0	100	1.04		62
				LC	4	5	5	12	31	1.0			LC	37
				SC	4	7	6	3	24	1.0			SC	23
3.13	C1	110	1.00	EW	91	86	89	88	41	0.6
				LC	0	6	5	6	23	0.5
				SC	5	4	2	2 8	17	0.8
	C2	109	0.98	EW	86	80	87	91	56	1.0	110	0.99		48	none
				LC	4	9	4	4	29	0.9			LC	26	none
				SC	6	7	5	1	26	1.1			SC	22	none
6.25	C1	125	1.23	EW	81	87	79	82	63	0.9
				LC	10	3	11	11	39	0.9
				SC	5	6	6	3	22	1.0
	C2	72	0.85	EW	87	85	86	87	63	1.1	98	1.04		63	1
				LC	7	7	4	6	38	1.2			LC	38	1
				SC	2	4	6	3	23	1.0			SC	23	none
12.5	C1	123	1.23	EW	86	88	88	86	40	0.6
				LC	3	3	4	6	17	0.4
				SC	7	5	4	4	22	1.0
	C2	114	1.01	EW	83	91	83	89	51	0.9	118	1.12		46	none
				LC	7	2	11	5	33	1.1			LC	25	none
				SC	6	3	2	2	17	0.7			SC	19	none
25	C1	111	1.03	EW	88	89	88	82	49	0.7
				LC	4	4	5	7	26	0.6
				SC	4	3	3	7	22	1.0
	C2	107	1.18	EW	85	86	86	82	53	0.9	109	1.11		51	none
				LC	9	9	3	10	36	1.1			LC	31	none
				SC	2	1	7	4	16	0.7			SC	19	none
50	C1	168	1.16	EW	86	85	89	87	44	0.6
				LC	6	9	5	5	29	0.7
				SC	4	2	2	4	14	0.6
	C2	123	1.18	EW	89	83	85	80	55	1.0	145	1.17		49	none
				LC	2	5	9	6	25	0.8			LC	27	none
				SC	5	8	2	10	28	1.2			SC	21	none
100	C1	131	1.33	EW	87	83	78	89	49	0.7
				LC	5	6	10	3	24	0.6
				SC	4	7	8	4	23	1.1
	C2	95	1.05	EW	84	85	87	88	53	0.9	113	1.19		51	none
				LC	7	5	8	2	28	0.9			LC	26	none
				SC	5	6	1	6	23	1.0			SC	23	0
NQO 0.1 µg /mL	C1	21	0.96	EW	24	27	26	26	682	10.1
				LC	50	35	40	33	275	6.3
				SC	22	34	30	37	200	9.1
	C2	19	0.76	EW	19	40	33	32	745	13.1	20	0.86		713	651
				LC	41	36	40	35	332	10.6			LC	303	266
				SC	36	20	23	29	218	9.1			SC	209	186

Adj. RTG: adjusted relative total growth                                         C1: culture 1

CE2: cloning efficiency                                                                  C2: culture 2

*: empty wells are counted on a total number of 96wells               0: vehicle control (water for injections)

MF: mutation frequency                                                                  NQO: 4--Nitroquinoline-N-oxide

IMF: Induced Mutation Frequency                                                  P: precipitate observed in the culture medium at the end of treatment         

SC: wells with small colonies

LC: wells with large colonies

EW: empty wells

 

Table3.Mutagenicity experiment with S9 mix, 3-hour treatment: mutagenicity results

Doses µg/mL		Adj. RTG %	CE2		Wells*				MFx10^-6	Ratio MF	Mean values of C1and C2
											Adj RTG%	CE2	MFx10^-6		IMF
0	C1	100	1.01	EW	83	83	77	76	92	1.0
				LC	9	6	15	13	59	1.0
				SC	4	7	5	7	31	1.0
	C2	100	0.69	EW	78	80	78	84	133	1.0	100	0.85		112
				LC	8	9	6	5	55	1.0			LC	57
				SC	10	7	12	7	72	1.0			SC	51
3.13	C1	82	0.96	EW	73	84	70	70	133	1.5
				LC	11	6	13	11	59	1.0
				SC	14	6	13	15	69	2.3
	C2	97	1.05	EW	83	73	65	70	133	1.0	89	1.01		133	21
				LC	8	8	15	15	61	1.1			LC	60	3
				SC	5	15	16	12	64	0.9			SC	67	15
6.25	C1	108	0.79	EW	78	80	76	76	135	1.5
				LC	13	9	11	15	84	1.4
				SC	6	7	9	5	46	1.5
	C2	106	0.95	EW	77	74	76	66	142	1.1	107	0.87		139	27
				LC	13	10	11	17	75	1.4			LC	80	23
				SC	6	12	10	14	61	0.9			SC	53	2
12.5	C1	117	1.14	EW	71	80	78	74	104	1.1
				LC	12	12	9	10	52	0.9
				SC	14	4	10	12	48	1.6
	C2	110	1.08	EW	73	75	73	80	113	0.8	114	1.11		108	none
				LC	13	11	13	7	56	1.0			LC	54	none
				SC	13	10	10	9	54	0.7			SC	51	none
25	C1	115	1.16	EW	70	76	70	78	115	1.3
				LC	13	14	18	11	68	1.2
				SC	13	8	11	8	47	1.6
	C2	119	0.94	EW	78	74	81	78	113	0.8	117	1.05		114	2
				LC	10	13	13	12	71	1.3			LC	70	13
				SC	8	10	2	6	37	0.5			SC	42	none
50	C1	106	1.16	EW	74	79	71	82	98	1.1
				LC	11	9	15	9	52	0.9
				SC	11	8	10	5	40	1.3
	C2	102	1.03	EW	74	75	78	68	128	1.0	104	1.09		113	1
				LC	9	12	10	15	62	1.1			LC	57	0
				SC	14	9	8	14	61	0.8			SC	50	none
100	C1	84	1.05	EW	74	81	74	68	123	1.3
				LC	9	15	14	22	81	1.4
				SC	13	10	9	7	51	1.7
	C2	125	0.96	EW	74	85	75	74	114	0.9	105	1.01		119	6
				LC	11	3	12	13	55	1.0			LC	68	11
				SC	11	8	9	9	52	0.7			SC	52	1
CPA 3µg/mL	C1	40	0.57	EW	30	20	26	17	1242	13.6
				LC	21	31	23	30	280	4.8
				SC	55	55	54	65	794	26.0
	C2	46	0.53	EW	17	31	24	18	1364	10.3	43	0.55		1303	1191
				LC	38	29	33	31	392	7.1			LC	336	279
				SC	56	49	49	54	734	10.2			SC	764	713

Adj. RTG: adjusted relative total growth                                          C1: culture 1

CE2: cloning efficiency                                                                   C2: culture 2

*: empty wells are counted on a total number of 96wells               0: vehicle control (water for injections)

MF: mutation frequency                                                                   CPA: Cyclophosphamide

IMF: Induced Mutation Frequency                                                  

P: precipitate observed in the culture medium at the end of treatment         

SC: wells with small colonies

LC: wells with large colonies

EW: empty wells

Conclusions:

Under the experimental conditions of this study, the test item, Saccharomyces cerevisiae, lysate, did notshow any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.

Executive summary:

The objective of this GLP compliant study was to evaluate the potential of the test item, Saccharomyces cerevisiae, lysate, to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK+/- mouse lymphoma cells according to OECD TG 490 method.

Material and methods

After a preliminary cytotoxicity test, the test item Saccharomyces cerevisiae, lysate, suspended in water for injections, for was tested in a first mutagenicity experiment, using 3-hour treatments, with and without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. In a second mutagenicity experiment, the test item was tested at the test site using a 24-hour treatment without metabolic activation system. Cultures of 20 mL at 5 x 105 cells/mL (3-hour treatments) or cultures of 50 mL at 2 x 105 cells/mL (24-hour treatment) were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% (3-hour treatment) or 10% (24-hour treatment) in a 37°C, 5% CO2 humidified incubator.

Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and cloning efficiency following the expression time (CE2). The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.

Results

The cloning efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria. For the positive control cultures, the increase in the mutation frequencies met the acceptance criteria. In addition, the upper limit of cytotoxicity observed in the positive control cultures had an Adj. RTG greater than 10%. The study was therefore considered to be valid. Since the test item was found non-cytotoxic but poorly soluble in the culture medium during the preliminary test, the highest dose level selection for the main experiment was based on the level of precipitate, according to the criteria specified in the international guidelines. The selected dose levels were 3.13, 6.25, 12.5, 25, 50 and 100 μg/mL for each experiment with or without S9 mix.

A precipitate was noted in the culture medium at dose levels ≥ 50 μg/mL at the end of the 3-hour treatment period and at dose levels ≥ 6.25 μg/mL at the end of the 24-hour treatment period.

No cytotoxicity was observed at any dose levels in any test conditions, as shown by the absence of any noteworthy decrease in the Adj. RTG.

Following the 3-hour treatments with and without S9 mix or the 24-hour treatment without S9 mix, no increase in the mutation frequency exceeding the GEF was observed at any of the tested dose levels relative to the corresponding vehicle control. Moreover, no dose-response relationship was demonstrated by the linear regression in any test conditions. These results did not meet the criteria for a positive response.

Conclusion

Under the experimental conditions of this study, the test item, Saccharomyces cerevisiae, lysate, did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Three in vitro key studies were available for mutagenicity evaluation for the registered substance:

An Ames study was performed (Orovecz, Klimisch 1, 2018, GLP, OECD TG 471). Bacteria strain Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA were treated with test substance in distilled water up to 5000 µg/plate with and without metabolic activation system. Three different test were performed as preliminary cytotoxicity test, initial mutation test (plate incorporation method) and confirmatory test (pre incubation method) and revertant colony number was counted. In these three different experiments, no cytotoxicity or mutagenicity were observed. Hence, the test substance was not considered as mutagenic for bacteria strain under the experimental conditions of the study.

An in vitro micronucleus assay was performed (Chevallier, Klimisch 1, 2018, GLP, OECD TG 487). Mammalian cell line L5178Y TK+/- was exposed to the test substance Saccharomyces Cerevisiae, lysate at concentrations up to 250 µg/mL, with and without metabolic activation system, during 3 or 24 hours period. After the treatment period, 2000 cells per dose were assessed for cytotoxicity (population doubling) and mutagenicity (micronucleus counted per cell). At the end of both 3- and 24-hour treatment periods, a precipitate was observed in the culture medium at dose levels >= 125 µg/mL. No cytotoxicity and no increase or micronucleated cells frequency were observed in the study. Hence, the test item did not induce any chromosome damage, or damage to the cell division apparatus for L5178Y TK +/- cells under the experimental conditions of the study.

An in vitro cytogenicity test was performed (Sire, Klimisch 1, 2018, GLP, OECD TG 480) on mammalian cellsz L5178 TK+/- cells. The cells were incubated with test substance at concentrations up to 1000 µg/mL with and without metabolic activation. Two incubation periods were used, 3- hours and 24- hours. Cytotoxicity was evaluated using Adjusted Relative Total Grown and Cloning Efficiency. The test item was found non-cytotoxic under the test conditions and no mutagenicity was observed under the experimental conditions of the study, after 3 hours of exposure.

Justification for classification or non-classification

Based on the three in vitro key studies (Orovecz, Klimisch 1, GLP, 2018, OECD TG 471; Chevallier, Klimisch 1, GLP, 2018, OECD 487 ; Sire, Klimisch 1, GLP, 2018, OECD TG 490), the test substance Saccharomyces Cerevisiae, lysate did not induce mutagenicity and clastogenicity on mammalian cells or bacteria strain. Hence, according to CLP critera, the test substance Saccharomyces Cerevisiae, lysate was not considered as mutagenic substance in vitro. Further in vivo evaluation was not required according to REACh regulation.