Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: gene mutation
Data waiving:
study technically not feasible
Justification for data waiving:
other:
Justification for type of information:
No indication of a postive result for 3 in vitro studies; bacterial reverse mutation (OECD 471), in vitro chromosome aberration (OECD 473), or in vitro gene mutation assay (OECD 490) with or without metabolic activation
Cross-referenceopen allclose all
Reason / purpose:
data waiving: supporting information
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant study conducted according to internationally recognised test methods.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Also compliant with Guideline for Screening Mutagenicity Testing of Chemicals Japan
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Lot/batch No.: C-120
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction (mixed induction rat liver preparation)
Test concentrations with justification for top dose:
Without S9 mix 0 313 625 1250 2500 &5000 ug/plate
With S9 mix 0 313 625 1250 2500 & 5000 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Substance is soluble in acetone.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
furylfuramide
Remarks:
TA100, TA98 in absence of S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA1535 in absence of S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
WP2 in absence of S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537 in absence of S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
All strains in the presence of S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Preincubation period: 10h
- Exposure duration: 48h at 37 degrees C
- Expression time (cells in growth medium): 48h
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: plates/test: 3

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: not observed at doses of up to 5000 ug/plate with or without S9

Evaluation criteria:
An increase in colony count of more than 2 fold when compared with concurrent controls and/or a dose dependent increase in the colony count which is statistically significantly higher than the count for the controls.
Statistics:
No statistical analyses were done.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
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 applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Evaporation from medium: No data
- Water solubility: N/A
- Precipitation: No
- Other confounding effects: No

RANGE-FINDING/SCREENING STUDIES: yes range of doses tested 1.22-5000ug/plate.

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxicity was not observed at 313-5000 ug/plate
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1 - Results of reverse mutation test I

With (+) or

Dose

(µg/plate)

Number of revertants Mean (±SD)

without(-)

Base-pair substitution type

Frameshift type

S9 mix

TA100

TA1535

WP2 uvrA

TA98

TA1537

 

0

105 (±3)

15 (±5)

34 (±4)

23 (±4)

9 (±2)

 

313

106 (±6)

17 (±4)

33 (±7)

23 (±4)

10 (±2)

S9 mix

625

113 (±7)

20 (±5)

38 (±2)

25 (±4)

8 (±1)

(-)

1250

124 (±16)

19 (±3)

38 (±6)

28 (±5)

10 (±1)

 

2500

123 (±12)

21 (±2)

36 (±6)

26 (±2)

8 (±2)

 

5000

116 (±9)

23 (±1)

36 (±3)

25 (±8)

8 (±3)

 

0

107 (±8)

16 (±1)

29 (±5)

35 (±3)

11 (±3)

 

313

114 (±8)

17 (±2)

35 (±2)

31 (±3)

13 (±3)

S9 mix

625

115 (±12)

20 (±2)

42 (±2)

35 (±2)

11 (±1)

(+)

1250

116 (±10)

20 (±1)

43 (±5)

39 (±3)

12 (±0)

 

2500

125 (±18)

21 (±3)

34 (±3)

36 (±3)

11 (±1)

 

5000

131 (±24)

21 (±3)

37 (±3)

39 (±3)

12 (±2)

+ve control

Chemical

AF-2

SA

ENNG

AF-2

9-AA

S9 mix(-)

Doseµg/plate

0.01

0.5

2

0.1

80

 

Colonies/plate

514 (±14)

441 (±30)

833 (± 42)

549 (±40)

313 (±6)

+ve control

Chemical

2-AA

2-AA

2-AA

2-AA

2-AA

S9 mix(+)

Doseµg/plate

1

2

10

0.5

2

 

Colonies/plate

1301 (±82)

228 (±3)

1092 (±28)

461 (±10)

189 (±13)

AF-2 = 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide; SA = sodium azide; ENNG = N-ethyl-n’-nitro-N-nitrosoguanidine;  9-AA = 9-aminoacridine; 2 -AA = 2 -aminoanthracene

 

Table 2 - Results of reverse mutation test II

With (+) or

Dose

(µg/plate)

Number of revertants Mean (±SD)

without(-)

Base-pair substitution type

Frameshift type

S9 mix

TA100

TA1535

WP2 uvrA

TA98

TA1537

 

0

151 (±5)

11 (±2)

33 (±2)

20 (±3)

8 (±2)

 

313

130 (±6)

10 (±1)

41 (±5)

18 (±2)

8 (±1)

S9 mix

625

135 (±3)

10 (±1)

39 (±2)

21 (±5)

7 (±2)

(-)

1250

135 (±13)

10 (±3)

39 (±7)

17 (±2)

9 (±3)

 

2500

142 (±6)

10 (±1)

42 (±1)

19 (±2)

11 (±1)

 

5000

140 (±20)

9 (±1)

41 (±1)

18 (±1)

8 (±1)

 

0

111 (±6)

10 (±1)

40 (±3)

28 (±2)

17 (±2)

 

313

135 (±13)

10 (±2)

48 (±3)

26 (±3)

13 (±2)

S9 mix

625

151 (±17)

10 (±1)

45 (±8)

29 (±5)

16 (±3)

(+)

1250

135 (±24)

12 (±1)

45 (±7)

30 (±4)

13 (±4)

 

2500

146 (±8)

10 (±1)

41 (±4)

31 (±10)

16 (±4)

 

5000

140 (±5)

12 (±2)

39 (±2)

29 (±3)

13 (±3)

+ve control

Chemical

AF-2

SA

ENNG

AF-2

9-AA

S9 mix(-)

Doseµg/plate

0.01

0.5

2

0.1

80

 

Colonies/plate

633 (±27)

485 (±8)

929 (±39)

531 (±53)

436 (±15)

+ve control

Chemical

2-AA

2-AA

2-AA

2-AA

2-AA

S9 mix(+)

Doseµg/plate

1

2

10

0.5

2

 

Colonies/plate

1230 (±38)

250 (±2)

1305 (±68)

510 (±19)

190 (±27)

 

AF-2 = 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide; SA = sodium azide; ENNG = N-ethyl-n’-nitro-N-nitrosoguanidine;  9-AA = 9-aminoacridine; 2 -AA = 2 -aminoanthracene

Conclusions:
Interpretation of results (migrated information):
negative

Based on results under the conditions of this test the substance was not considered to be mutagenic to Salmonella typhimurium TA100, TA 1535, TA98, TA1537 & E coli WP2 uvrA.
Executive summary:

A bacterial reverse mutation assay (Ames test) has been undertaken following OECD test methods.

The substance does not induce reverse mutation in Salmonella typhimurium or Escherichia coli.

Reason / purpose:
data waiving: supporting information
Reference
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant study conducted in accordance with internationally recognised test methods
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
not specified
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Lot/batch No.: C-120
Target gene:
N/A
Species / strain / cell type:
other: Chinese hamster lung cells CHL/IU
Details on mammalian cell type (if applicable):
- Type and identity of media: Inactivated calf serum in Eagle MEM
- Properly maintained: No data
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction (mixed function induction of rat liver)
Test concentrations with justification for top dose:
-S9 (continuous treatment): 0, 1.3, 2.5, 5.0 mg/ml
-S9 (short term treatment): 0, 1.3, 2.5, 5.0 mg/ml
+S9 (short term treatment): 0, 1.3, 2.5, 5.0 mg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone; supplied by Wako Pure Chemical Industries Co.
- Justification for choice of solvent/vehicle: Substance is poorly soluble in water; very soluble in acetone & DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
-S9; 6 & 24 hours of exposure
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
+S9; 6 hours exposure
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Preincubation period:
- Exposure duration: For continuous treatment, cells were treated for 24 hours in the absence of S9; for short-term treatment cells were treated for 6 hours with & without S9 and cultivated with fresh media for 18 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcimid 0.1 ug/mL
STAIN (for cytogenetic assays): 3% Giemsa solution

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 metaphases/slide of each replicate

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No cytotoxicity up to and including 5.0 mg/mL when inhibition of cell growth determined.

OTHER EXAMINATIONS:
- Determination of polyploidy:Negative
- Determination of endoreplication: No data or negative if equivalent to clastogenicity
Evaluation criteria:
A dose related increase or a reproducible increase in the number of cells with chromosome aberrations.
An increase in the number of polyploid cells.
An increase in the number of cells with endoreduplicated chromosomes
Statistics:
No data, thought to be Fisher's Exact Test
Species / strain:
other: Chinese hamster lung cells (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
5 mg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Evaporation from medium: No
- Water solubility: poor
- Precipitation: No
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES: yes; growth inhibition test (range of concentrations: 0.0-5.0 mg/mL

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxicity observed
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1 - Chromosome analysis of cells following short-term treatment with and without S9 mix

 

Group

Concentration (μg/mL)

S9

Exposure Time

(hours)

Cell

No. of cells

analysed

No. of structural aberrations

Number of cells with aberrations (%)

Polyploidy

mix

growth index

gap

ctb

cte

csb

cse

frg

Total

(%)

Solvent control

0

-

6

100

200

0

0

0

1

0

0

1

1 (0.5)

0.0

 

1250

-

6

107

200

0

1

0

0

0

0

1

1 (0.5)

0.0

Test substance

2500

-

6

111

200

0

0

0

1

0

0

1

1 (0.5)

0.0

 

5000

-

6

107

200

0

1

0

2

0

0

3

3 (1.5)

0.0

MMC

0.1

-

6

113

200

4

17

76

1

2

0

96

91 (45.5)

0.0

 

Solvent control

 

0

 

+

 

6

 

100

 

200

 

1

 

1

 

1

 

2

 

0

 

0

 

4

 

3 (1.5)

 

0.0

TOTM

1250

+

6

105

200

0

2

0

1

2

0

5

5 (2.5)

0.0

T0TM

2500

+

6

103

200

0

0

0

1

1

0

2

2 (1.0)

0.0

TOTM

5000

+

6

94

200

0

1

1

0

0

0

2

2 (1.0)

0.0

BP

20

+

6

133

200

2

14

144

0

2

0

160

147 (73.5)

0.0

Abbreviations:

gap: chromatid gap and chromosome gap, ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange (dicentric and ring etc.),frg: fragment,MMC: mitomycin C,BP: benzo(a)pyrene

 

 

Table 2 - Chromosome analysis of cells following continuous treatment without S9 mix

  

Group

Concentration (μg/mL)

S9

Exposure Time

(hours)

Cell

No. of cells

analysed

No. of structural aberrations

Number of cells with aberrations (%)

Polyploidy

mix

growth index

gap

ctb

cte

csb

cse

frg

Total

(%)

Solvent control

0

-

24

100

200

1

0

1

1

0

0

2

2 (1.0)

0.0

 

1250

-

24

98

200

1

0

0

0

0

0

0

0 (0.0)

0.0

Test substance

2500

-

24

104

200

0

0

1

1

0

0

2

2 (1.0)

0.0

 

5000

-

24

107

200

2

1

0

0

0

0

1

1 (0.5)

0.0

MMC

0.03

-

24

103

200

3

20

38

2

2

0

62

61 (30.5)

0.0

 

Abbreviations:

gap: chromatid gap and chromosome gap, ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange (dicentric and ring etc.),frg: fragment,MMC: mitomycin C

Conclusions:
Interpretation of results (migrated information):
negative

In the conditions in which this study was performed the tested substance, at doses of up to 5.0 mg/mL, did not induce chromosome aberrations with or without S9 in cultured mammalian somatic cells.
Executive summary:

The substance has been assayed for its ability to cause chromosomal damage in cultured mammalian cells following in-vitro treatment in the absence and presence of S9 metabolic activation.

The substance did not induce chromosomal aberrations in human lymphocytes after in-vitro treatment

Reason / purpose:
data waiving: supporting information
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2017-04-13 to 2017-06-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
Adopted June 2016
Deviations:
no
GLP compliance:
yes
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: Polynt Lot No. 3606217009
- Expiration date of the lot/batch: 09 Jan 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient conditions
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: Soluble/miscible and stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Rat S-9 tissue homogenate
Test concentrations with justification for top dose:
0.125, 0.250, 0.500, 1.00 and 2.00 microlitres/mL
Highest concentration examined is that indicated by the test guideline
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Commonly used vehicle for testing of poorly water soluble/miscible sustances
Untreated negative controls:
yes
Remarks:
Solvent
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
methylmethanesulfonate
Details on test system and experimental conditions:
S9 tissue homogenate
S9 tissue homogenate was prepared from the livers of five young male Sprague-Dawley rats which had received treatment with phenobarbital and betanaphthoflavone to induce high levels of xenobiotic
metabolising enzymes.

Preparation of test cell cultures
A cell suspension (1E6 cells/mL) in complete medium was prepared. The cultures were incubated at 37°C. At the end of the incubation period, the treatment medium was removed and the cultures centrifuged and washed with Phosphate Buffered Saline (PBS).

Cytotoxicity assay
A preliminary cytotoxicity test was performed in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined.
Treatments were performed in the absence and in the presence of S9 metabolic activation for 3 hours and for 24 hours only in the absence of S9 metabolic activation. A single culture was used at each test point. After washing in PBS, cells were resuspended in 20 mL RPMI minimal medium. Cell concentrations were adjusted to 8 cells/mL using complete medium and, for each dose level, 0.2 mL was plated into 96 microtitre wells. The plates were incubated at 37°C in a 5% CO2 atmosphere
(100% nominal relative humidity) for 8 days. Wells containing viable clones were identified by eye using background illumination and then counted.

Mutation assay
Treatment of cell cultures
Experiments were performed including vehicle and positive controls, in the absence and presence of S9 metabolising system.
Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture.
In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours). Since negative results were obtained in this experiment without metabolic activation, the second experiment in the absence of S9 metabolism, was performed using a long treatment time (24 hours). After washing in PBS, cells were resuspended in fresh complete medium (10%) and cell densities were determined. The number of cells was adjusted to give 2E5 cells/mL. The cultures were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.

Determination of survival
Following adjustment of the cell densities, samples of the cultures were diluted to 8 cell/ml using complete medium (20%). A 0.2 mL aliquot of each diluted culture was placed into each well of two 96-well plates. The plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 8 days. After incubation, wells containing viable clones were identified by eye using background illumination and then counted.

Expression period
During the expression period (two days after treatment) the cell populations were subcultured in order to maintain them in exponential growth. At the end of this period the cell densities of each culture were determined and adjusted to give 2E5 cells/mL.

Plating for 5-trifluorothymidine resistance
After dilution, the cell suspensions in complete medium B (20%) were supplemented with trifluorothymidine and an estimated 2E3 cells were plated in each well of four 96-well plates. Plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified . In addition, the number of wells containing large colonies and the number containing small colonies were scored.

Plating for viability
After dilution, in complete medium (20%), an estimated 1.6 cells/well were plated in each well of two 96-well plates. These plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified as above and counted.
Evaluation criteria:
The assay was considered valid if the following criteria were met:
(i) The cloning efficiencies at Day 2 in the untreated/solvent control cultures fell within the range of 65-120%.
(ii) The untreated/solvent control suspension growth over 2 days fell within the range of 8-32 (3 hours exposure or 32-180 (24 hours exposure).
(iii) The mutant frequencies in the untreated/solvent control cultures fell within the range of 50E-6 - 170E-6 viable cells.
(iv) The positive control chemicals induced a clear increase in mutant frequency above the spontaneous background with at least 40% of the induced mutant frequency reflected in the small colony mutation frequency

The assay was considered positive if:
(i) The induced mutation frequency was higher than teh global evaluation factor suggested for the assay
(ii) There was a significant dose-relationship as indicated by linear trend analysis
Statistics:
Statistical analysis was performed according to UKEMS guidelines (Robinson W.D., 1990).
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

In Mutation assay I (3 hours exposure), slight precipitation was noted upon addition of the highest concentration of the test item to the cultures both in the absence and presence of S9 metabolism. No precipitation was observed in Mutation assay II (24 hours exposure).

Solvent and positive control cultures were included in each mutation experiment and mutant frequencies in the solvent control cultures fell within the normal range. Both positive control items resulted in an increase in the small colony mutant frequency above that seen in the solvent control.

The cloning efficiencies at Day 2 in the negative control cultures fell within the range of 65 -120%. The control suspension growth over 2 days fell within the range of 8 - 32 for 3 hour treatments and 32 - 180 for 24 hour treatment.

Mild toxicity was observed at the intermediate dose levels in the 3 hour treatment series in the absence of S9 metabolic activation. No relevant toxicity was observed at any concentration tested, with the remaining treatment conditions.

In all experimental conditions examined, no increase in mutation frequency above the concurrent negative control value exceeded the Global Evaluation Factor.

In Main Assay I (3 hours exposure), in the absence of S9 metabolic activation, higher than usual heterogeneity was observed between replicate cultures for mutation at 1.00 µL/mL, therefore this concentration

has been excluded from the statistical analysis. This finding did not affect the validity of the test.

No statistically significant dose-effect relationship was observed at any treatment time, in the absence or presence of S9 metabolism.

The test substance did not have any obvious effect on the osmolality or pH of the treatment medium.

Conclusions:
The tested substance does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation.
Executive summary:

The substance has been examined for mutagenic activity by assaying for the induction of 5 -trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the

absence and presence of S9 metabolic activation, using a fluctuation method.

The substance does not induce mutation in mouse lymphoma L5178Y cells after in vitro treatment in the absence or presence of S9 metabolic activation.

Data source

Materials and methods

Results and discussion

Applicant's summary and conclusion