Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No gene or chromosome mutation found in any of the tests.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June - August 2017
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
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction
Test concentrations with justification for top dose:
Pre-experiment
The toxicity of the test item was determined with tester strains TA 98 and TA 100 in a pre-experiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test).
Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.
The test item was tested in the pre-experiment with the following concentrations:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Main experiment
The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 5000 µg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. Two independent experiments were performed with the following concentrations: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

All concentrations used - per active ingredient (90%, conversion factor 1.1)
Vehicle / solvent:
Aqua destillata
Untreated negative controls:
yes
Remarks:
A. dest., Eurofins Munich, Lot No. 170619, 170627,170710
Negative solvent / vehicle controls:
yes
Remarks:
A. dest., Eurofins Munich, Lot No. 170619, 170627,170710
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 1. 4-nitro-o-phenylene-diamine; 2. 2-aminoanthracene
Details on test system and experimental conditions:
Bacteria
Five strains of S. typhimurium with the following characteristics were used:
TA 98: his D 3052; rfa-; uvrB-; R-factor: frame shift mutations
TA 100: his G 46; rfa-; uvrB-; R-factor: base-pair substitutions
TA 1535: his G 46; rfa-; uvrB-: base-pair substitutions
TA 1537: his C 3076; rfa-; uvrB-: frame shift mutations
TA 102: his G 428 (pAQ1); rfa-; R-factor: base-pair substitutions

Tester strains TA 98, TA 1535 and TA 102 were obtained from MOLTOX, INC., NC 28607, USA. Tester strains TA 100 and TA 1537 were obtained from Xenometrix AG, Switzerland. They were stored as stock cultures in ampoules with nutrient broth (OXOID) supplemented with DMSO (approx. 8% v/v) over liquid nitrogen.

Samples of each tester strain were grown by culturing for 12 h at 37 °C in Nutrient Broth to the late exponential or early stationary phase of growth (approx. 109 cells/mL). A solution of 125 µL ampicillin (10 mg/mL) (TA 98, TA 100, TA 102) was added in order to retain the phenotypic characteristics of the strain.

S9 Homogenate
The S9 liver microsomal fraction was prepared at Eurofins Munich and obtained from Trinova Biochem GmbH, Giessen, Germany. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route (Eurofins Munich) and male Sprague Dawley rats were induced with phenobarbital / β-naphthoflavone (Trinova).

The following quality control determinations were performed by Eurofins Munich:
a) Biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene
b) Sterility Test
A stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4 mL and stored at <= -75 °C.
The protein concentration in the S9 preparation was 34.2 mg/mL.

The following quality control determinations were performed by Trinova Biochem GmbH:
a) Alkoxyresorufin-0-dealkylase activities
b) Test for the presence of adventitious agents
c) Promutagen activation (including biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene)
A stock of the supernatant containing the microsomes is frozen in aliquots of 5 mL and stored at <= -75 °C.
The protein concentration in the S9 preparation was 33.7 mg/mL.
Evaluation criteria:
Evaluation of cytotoxicity
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "N" or "B", respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

Evaluation of mutagenicity
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
During the described mutagenicity test and under the experimental conditions reported, the substance did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, it is considered to be non-mutagenic in this bacterial reverse mutation assay.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 2017 - April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Human peripheral blood lymphocytes from healthy and non-smoking donors with no known recent exposure to genotoxic chemicals and radiation were used to examine the ability of chemicals to induce cytogenetic damage and thus to identify potential carcinogens or mutagens in vitro. For this study (in each experiment) blood was collected only from a single donor to reduce inter-individual variability.
Blood samples were drawn by venous puncture and collected in heparinized tubes. Before use the blood was stored under sterile conditions at 4 °C for a maximum of 4 h. Whole blood samples treated with an anti-coagulant (e. g. heparin) were pre-cultured in the presence of mitogen (phyto-haematogglutinin, PHA).
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Concentrations in the main experiment were selected based on the prelimnary experiment, where the following concentrations were tested: 5, 10, 25, 50, 100, 250, 500, 1000, 2500, 5000 ug/mL
Precipitation was observed at concentrations 1000 ug/mL and higher. Strong cytotoxicity was observed at 250 ug/mL

Main experiment:
Experiment I
without S9 mix: 1, 1.25, 5, 10, 25, 50, 100, 150, 200 ug/mL (microscopic analysis: 10, 25, 50 ug/mL)
with S9 mix: 2, 5, 10, 25, 50, 100, 150, 200, 250 ug/mL (microscopic analysis 10, 25, 50, 100 ug/mL)

Experiment II
without S9 mix: 10, 25, 50, 100, 150, 200, 250, 300, 500 ug/mL (microscopic analysis: 50, 100, 150 ug/mL)

All concentrations used - per active ingredient (90%, conversion factor 1.1)
Vehicle / solvent:
RPMI cell culture medium
Untreated negative controls:
yes
Remarks:
treatment medium (complete culture medium without FBS)
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
Complete Culture Medium:
RPMI 1640 medium supplemented with:
15 % fetal bovine serum (FBS)
100 U/100 µg/mL penicillin/streptomycin solution
0.24 g/mL PHA-L
Also used for the long-term treatment and the post incubation.

Treatment Medium (short-term exposure):
Complete culture medium without FBS.

All incubations were done at 37 °C in humidified atmosphere with 5% CO2.

Treatment:
Experiment I: Short-term exposure 4 h (without and with S9 mix)
After 48 h the culture medium was replaced with serum-free medium containing the test item (without metabolic activation) and serum-free medium containing the test item with 50 µL/mL S9 mix (with metabolic activation). After 4 h the cells were spun down by gentle centrifugation for 10 min. The supernatant with the dissolved test item was discarded and the cells were resuspended in PBS. The washing procedure was repeated once as described. After washing, the cells were resuspended in complete cell culture medium. The cells were prepared 24 h after the beginning of the treatment.
 Experiment II: Long-term exposure 24 h (without S9 mix):
After 48 h the culture medium was replaced with complete medium (with 15% FBS) containing the test item without S9 mix. The treated cells were prepared at the end of the treatment.

For all experiments, the effect of the test item on cell cycle progression was investigated by addition of BrdU to the cultures. For each experiment the solvent and negative control and the highest dose group were evaluated to reassure the replication time of the cultured lymphocytes.

At least 2 h before harvesting, colcemid was added to the cultures (final concentration 0.2 µg/mL). The cultures were harvested by centrifugation 24 h after beginning of treatment. The supernatant was discarded and the cells were resuspended in approximately 5 mL hypotonic solution (0.4% KCl). The cell suspension was incubated at room temperature for 20 min. After removal of the hypotonic solution by centrifugation the cells were fixed with 3+1 methanol + glacial acetic acid. The fixation procedure was repeated twice. Slides were prepared by dropping the cell suspension onto a clean microscopic slide.The cells were stained with giemsa and according to the Fluorescent plus Giemsa technique, respectively.The slides were coverslipped using 2-3 drops of Eukitt(R). Afterwards they were air dried.

Analysis of metaphase cells:
All slides, including those of positive and negative controls were independently coded before microscopic analysis. Evaluation of the cultures was performed using microscopes with 100x oil immersion objectives. As structural chromosomal aberrations breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded. Gaps were recorded as well but not included in the calculation of the aberration rates. The definition of a gap was as follows: an achromatic region (occurring in one or both chromatids) independent of its width. The remaining visible chromosome regions should not be dislocated neither longitudinally nor laterally. At least, if available, 300 well spread metaphases per concentration and validity controls were scored for cytogenetic damage The number of metaphases scored can be reduced when high numbers of cells with chromosome aberration were observed and the chemical was considered as clearly positive. Metaphases with 46±2 centromeres regions were included in the analysis.
To describe a cytotoxic effect the mitotic index (% cells in mitosis; by counting the number of mitotic cells in 1000 cells) was determined. Additionally the number of polyploid cells was scored. Polyploid means a near tetraploid karyotype in the case of this aneuploid cell line.
Rationale for test conditions:
Guideline study
Evaluation criteria:
Evaluation of results:
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the 95% control limits of the historical negative control data.

When all of these criteria are met, the test chemical is then considered able to induce chromosomal aberrations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the 95% control limits of the historical negative control data.

The test chemical is then considered unable to induce chromosomal aberrations in cultured human peripheral blood lymphocyte cells in this test system.
Statistics:
Biometry - Statistical significance at the 5% level (p < 0.05) was evaluated by the Fischer´s exact test. The p value was used as a limit in judging for significance levels in comparison with the corresponding negative control. Aberrant cells without gaps were only used for the calculation. Gaps are recorded separately and reported but generally not included in the total aberration frequency calculation according to the guideline.
Biometry - trend test - Statistical significance at the 5% level (p < 0.05) was evaluated by the ² test for trend. The p value was used as a limit in judging for significance levels.
Key result
Species / strain:
lymphocytes: Human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid

Summary: Experiment I, without and with metabolic activation

Dose group

Concen-tration [µg/mL]

Relative Mitotic Index [%]

Proliferation Index

Mean % Aberrant Cells

Historical Laboratory Negative Control Range

Precipi-tation (+/-)

Statistics

incl. Gaps

excl. Gaps

without

4 h treatment,

24 h preparation interval

C

0

100

1.35

3.7

2.7

-0.09 % – 3.47 % aberrant cells excl. gaps 

-

/

4

10

40

/

4.7

2.6

-

-

5

25

60

/

5.0

4.7

-

-

6

50

53

1.20

3.7

2.0

-

-

EMS

600

86

/

14.2

13.3

 

-

+

 

with4 h treatment,

24 h preparation interval

C

0

100

1.37

2.3

1.3

-0.41 % – 3.42 % aberrant cells escl. gaps 

-

/

3

10

107

/

4.0

1.3

-

-

4

25

64

/

0.7

0.3

-

-

5

50

74

/

1.7

0.3

-

-

6

100

42

1.45

2.6

2.6

-

-

CPA

5

42

/

12.6

10.5

 

-

+

 

The mitotic index was determined in 1000 cells per culture of each test group. The relative values of the mitotic index are related to the negative controls.

 C: Negative Control (Culture Medium)

EMS: Ethylmethanesulfonate

CPA: Cyclophosphamide

a: - without precipitation. + with precipitation

b: statistical significant increase compared to negative controls (Fisher’s exact test, p< 0.05), +: significant; -not significant

Summary: Experiment II, without metabolic activation 

Dose group

Concen-tration [µg/mL]

Relative Mitotic Index [%]

Proliferation Index

Mean % Aberrant Cells

Historical Laboratory Negative Control Range

Precipi-tation (+/-)

Statistics

incl. Gaps

excl. Gaps

without

24 h treatment,

24 h preparation interval

C

0

100

/

4.3

2.7

-0.57 % - 3.16 % aberrant cells excl. gaps 

-

/

3

50

100

/

4.0

1.7

-

-

4

100

65

/

4.3

2.7

-

-

5

150

52

/

6.0

3.0

-

-

EMS

400

47

/

31.0

29.0

 

-

+

 

The mitotic index was determined in 1000 cells per culture of each test group. The relative values of the mitotic index are related to the negative controls.

 

C: Negative Control (Culture Medium)

EMS: Ethylmethanesulfonate

a: - without precipitation. + with precipitation

b: statistical significant increase compared to negative controls (Fishers exact test, p< 0.05), +: significant; -not significant

Conclusions:
It can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocyte cells.
Therefore, the substance is considered to be non-clastogenic in this chromosome aberration test.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June-November 2017
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 July 29, 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial forward mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Remarks:
thymidine kinase locus
Details on mammalian cell type (if applicable):
clone TK+/- -3.7.2C
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 Homogenate
Test concentrations with justification for top dose:
The selection of the concentrations used in the main experiment was based on data from the pre-experiment. The test item was investigated at the following concentrations:

without metabolic activation:
60, 70, 75, 80, 85, 86, 87, µg/mL

and with metabolic activation:
104, 108, 112, 115, 118, 121, 124 µg/mL

All concentrations used - per active ingredient (90%, conversion factor 1.1)
Vehicle / solvent:
RPMI cell culture medium (RPMI + 5% HS)
Untreated negative controls:
yes
Remarks:
treatment medium
Negative solvent / vehicle controls:
yes
Remarks:
the same as negative control
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
methylmethanesulfonate
Details on test system and experimental conditions:
For a short-term exposure experiment 1 x 10^7 cells were suspended in 11 mL RPMI medium with 5% horse serum (25 cm2 flasks) and exposed to designated concentrations of the test item either in the presence or absence of metabolic activation in the mutation experiment. After 4 h the test item was removed by centrifugation (200 x g, 7 min) and the cells were washed twice with PBS. Subsequently the cells were suspended in 30 mL complete culture medium and incubated for an expression and growth period of 2 days in total at 37 °C in 5% CO2/95% humidified air. The cell density was determined each day and adjusted to 3 x 10^5 cells/mL in a total culture volume of 20 mL, if necessary.
After the expression period the cloning efficiency (CE) of the cells was determined by seeding a statistical number of 1.6 cells/well in two 96-well plates. The cells were incubated for at least 6 days at 37 °C in a humidified atmosphere with 5% CO2. Analysis of the results was based on the number of cultures with cell growth (positive wells) and those without cell growth (negative wells) compared to the total number of cultures seeded. Additionally, cultures were seeded in selective medium. Cells from each experimental group were seeded in four 96-well plates at a density of approximately 2000 cells/well in 200 µL selective medium with TFT. The plates were scored after an incubation period of about 12 days at 37 °C in 5% CO2/95% humidified air.
Rationale for test conditions:
According to the OECD 490 Guideline.
Evaluation criteria:
Acceptability of the assay:
A mutation assay is considered acceptable if it meets the criteria mentioned in current international guidelines and the current recommendations of the IWGT:
- At least three out of four 96-well plates from the TFT resistance-testing portion of the experiment are scorable
- The cloning efficiency of the negative and/or solvent controls is in the range 65% -120%.
- The spontaneous mutant frequency in the negative and/or solvent controls is in the range 50-170 mutants per 106 cells
- The cell number of the negative/solvent controls should undergo 8-32 fold increase during a 2 day growth period (short-term treatment)
- The clastogenic positive controls (MMS and B[a]P) have to produce an induced mutant frequency (total mutant frequency minus concurrent negative control mutant frequency) of at least 300 mutants per 106 cells with at least 40% of the colonies being small colonies or with an induced small colony mutant frequency of at least 150 mutants per 106 cells The RTG must be greater than 10%.

Evaluation of results:
The test item is considered mutagenic if the following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 106 cells and
- a dose-dependent increase in mutant frequency is detected.
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation of the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative.
Statistics:
The mutant frequency was calculated by dividing the number of TFT resistant colonies by the number of cells plated for selection, corrected for the plating efficiency of cells from the same culture grown in the absence of TFT. For the microwell method used the Poisson distribution was used to calculate the plating efficiencies for cells cloned without and with TFT selection. Based on the null hypothesis of the Poisson distribution, the probable number of clones/well (P) is equal to
–ln(negative wells/total wells) and the plating efficiency (PE) equals P/(number of cells plated per well). Mutant frequency then was calculated as MF = (PE(cultures in selective medium)/PE(cultures in non-selective medium)). The mutant frequency is usually expressed as “mutants per 106 viable cells”.

Suspension growth (SG) of the cell cultures reflects the number of times the cell number increases from the starting cell density. When carrying out a short-term treatment (4 h) a 2-day growth period was considered. The relative total growth (RTG) is the product of the relative suspension growth (RSG; calculated by comparing the SG of the dose groups with the SG of the control) and the relative cloning efficiency (RCE) for each culture: RTG = RSG x RCE /100.
The mutant frequencies obtained from the experiments were compared with the Global Evaluation Factor (GEF). To arrive at a GEF, the workgroup (IWGT MLA Workgroup) analyzed distributions of negative/vehicle mutant frequencies of the MLA that they gathered from ten laboratories. The GEF is defined as the mean of the negative/vehicle mutant frequency plus one standard deviation. Applying this definition to the collected data, the GEF arrived to be 126 for the microwell method.
The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the solvent/negative controls were used as reference.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Summary results, without and with metabolic activation

 

Test Group

Conc.

 [µg/mL]

 RCEa[%]

RTGb[%]

MFc[mutants/ 106cells]

IMFd[mutants/ 106cells]

GEFeexceeded

Statistical

Significant Increasef

Precipitate

Exp

without

S9

 

C1

0

100.0

100.0

55.2

/

/

/

-

C2

/

/

/

-

2

60

103.4

115.6

67.1

11.9

-

-

-

3

70

100.0

93.0

55.4

0.2

-

-

-

4

75

105.1

73.0

53.8

-1.4

-

-

-

5

80

105.1

49.6

51.1

-4.1

-

-

-

10

85

110.7

18.2

52.5

-2.7

-

-

-

11

86

110.7

23.9

47.3

-7.9

-

-

-

12

87

110.7

14.5

58.8

3.6

-

-

-

EMS

300

62.1

54.6

1048.7

993.5

+

+

-

MMS

10

66.7

58.5

554.7

499.5

+

+

-

 

Exp

with S9

 

C1

0

100.0

100.0

55.4

/

/

/

 

C2

/

/

/

 

4

104

99.8

109.2

44.5

-10.9

-

-

 

5

108

93.8

76.0

44.3

-11.1

-

-

 

6

112

110.2

88.4

34.0

-21.5

-

-

 

7

115

110.2

59.9

38.9

-16.5

-

-

 

8

118

106.5

50.9

41.7

-13.7

-

-

 

9

121

93.8

30.6

38.1

-17.3

-

-

 

10

124

106.5

12.3

49.9

-5.5

-

-

 

B[a]P

1.5

86.9

60.4

455.8

400.4

+

+

 

C:       Negative Controls

a:        Relative Cloning Efficiency, RCE = [(CEdose group/ CEof corresponding controls) x 100]

            Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100)

b:        Relative Total Growth, RTG = (RSG x RCE)/100

c:        Mutant Frequency,

            MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800

d:        Induced Mutant Frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls

e:        Global Evaluation Factor, GEF (126); +: GEF exceeded, -: GEF not exceeded

f:         statistical significant increase in mutant frequency compared to negative controls (Mann Whitney test , p<0.05).
+: significant; -not significant

EMS:   Ethylmethanesulfonate

MMS:  Methylmethanesulfonate

B[a]P:  Benzo[a]pyrene

Conclusions:
In the described mutagenicity test under the experimental conditions reported, the test item is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

No positive results in any of the performed gene/chromosome mutation tests.