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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
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

Test material

Constituent 1
Reference substance name:
Saccharomyces cerevisiae, lysate
EC Number:
305-230-8
EC Name:
Saccharomyces cerevisiae, lysate
Cas Number:
94350-12-6
IUPAC Name:
Saccharomyces cerevisiae, lysate
Test material form:
solid: particulate/powder
Remarks:
light beige
Details on 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
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

Method

Target gene:
micronuclei
Species / strain
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.
Controls
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.

Results and discussion

Test results
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

Any other information on results incl. tables

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

 

 

Treatment

Cell concentration

used for treatment ( x 104cells/mL)

 

Culture

Post-treatment cell count

(x 104cells/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 104cells/mL)

 

Culture

Post-treatment cell count

(x 104cells/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 104cells/mL)

 

Culture

Post-treatment cell count

(x 104cells/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

 

 

Applicant's summary and conclusion

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.