Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three key in vitro genetic toxicity studies are available.

- the test substance was tested in the bacterial reverse mutation assay (Ames test) in five bacterial strains (S typhimurium T1535, TA98, TA100, TA1537 and E. Coli WP2 uvrA). The test substance was considered not mutagenic under the conditions of the test.

- the in vitro mammalian chromosome aberration test with the test substance was performed with human lymphocytes. The test item was shown to be non-clastogenic.

- a mouse lymphoma assay was conducted according to the OECD guideline 476 and EU method B.17. The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

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:
From February 28, 2000 to March 25, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
-an allowance for test material purity was made prior to each days formulation.
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 liver microsomal fraction
Test concentrations with justification for top dose:
Experiment 1:
5, 15, 50, 150, 500 and 1500 µg/plate for the strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
15, 50, 150, 500, 1500 and 5000 µg/plate for E. coli WP2 uvr A

Experiment 2:
5, 15, 50, 150, 500 and 1500 µg/plate for the strains S. typhimurium TA 1535, TA 98 and TA 100
50, 150, 500, 1500 and 5000 µg/plate for E. coli WP2 uvr A
15, 50, 100, 150, 200 and 500 µg/plate for the strain S. typhimurium TA 1537

Experiment 3:
15, 50, 100, 150, 200 and 500 µg/plate for the strain S. typhimurium TA 1537
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl formamide

Negative solvent / vehicle controls:
yes
Remarks:
(Dimethyl formamide)
Positive controls:
yes
Remarks:
(without metabolic activation)
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
3 µg/plate for TA100, 5 µg/plate for TA1535 and 2 µg/plate for WP2uvrA
Positive controls:
yes
Remarks:
(without metabolic activation)
Positive control substance:
9-aminoacridine
Remarks:
80 µg/plate for TA1537
Positive controls:
yes
Remarks:
(without metabolic activation)
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.2 µg/plate for TA98
Positive controls:
yes
Remarks:
(with metabolic activation)
Positive control substance:
other: 2-aminoanthracene
Remarks:
1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, 10 µg/plate for WP uvrA
Positive controls:
yes
Remarks:
(with metabolic activation)
Positive control substance:
benzo(a)pyrene
Remarks:
5 µg/plate for TA98
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth.

Evaluation criteria:
The test material was considered positive in this test system if there is a reproducible dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.
Statistics:
Dunnett's method of linear regression
Species / strain:
other: E. coli WP2 uvr A, TA1535, TA1537, TA98, TA100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at and above 500 µg/plate)
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: E. coli WP2 uvr A, TA1535, TA1537, TA98, TA100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at and above 500 µg/plate)
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation of the test substance was noted at the dose levels tested.

RANGE-FINDING/SCREENING STUDIES:
Preliminary toxicity study was performed with strains TA100 and WPuvrA with and without metabolic activation. Ten concentrations of the test material and a vehicle control (dimethyl formamide) were tested. The dose range of the test material was: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, and 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
All tester strain cultures exhibit a characteristic number, within the range of historical data, of spontaneous revertants per plate in the vehicle and untreated controls.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Experiment 1

 

Table 1: without metabolic activation

With or without S9-Mix

Test substance concentration µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

  

TA100             TA1535        WP2uvrA

Frameshift type

   

    TA98         TA1537

-

0

146

10.1 #

22

2.9

19

3.1

20

6.1

9

0.6

-

5

144

12.5

20

3.1

 

N/T

20

3.8

10

1.5

-

15

145

11.4

14

4.2

23

3.8

25

2.3

14

4.4

-

50

143

7.6

12

2.1

16

7.0

28

8.4

16*

3.8

-

150

112

4.0

13

2.3

24

3.1

25

4.0

18$$$

2.3

-

500

23

3.2

3

2.0

22

9.3

6

2.0

3V

1.7

-

1500

0V

0.0

0V

0.0

20

3.5

0V

0.0

0T

0.0

-

5000

N/T

N/T

0V

0.0

N/T

N/T

Positive controls

Name

ENNG

ENNG

ENNG

4NQO

9AA

S9-Mix

Concentration

(µg/plate)

3

5

2

0.2

80

 

Nº Colonies per plate

425

56.4

372

1.5

615

77.6

111

12.3

807

226.2

 

ENNG  N-ethyl-N'-nitro-N-nilrosoguanidine N/T  Not tested at this dose level

4NQO  4-Nitroquinoline-l-oxide                                   S  Sparse bacterial background lawn

9AA    9-Aminoacridine                                           T  Toxic, no bacterial background lawn

V        Very weak bacterial background lawn            $$$ p¿0.005

#         Standard deviation                                         *    p¿ 0,05         

 

 Table 2: with metabolic activation 

With or without S9-Mix

Test substance concentration (µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

 

TA100           TA1535      WP2uvrA

Frameshift type

 

  TA98               TA1537

+

0

151

18.6#

13

2.1

22

0.0

24

3.6

7

1.0

+

5

174

51.0

14

1.5

N/T

21

3.2

12

2.0

+

15

132

13

11

5.1

22

1.2

29

2.5

9

3.1

+

50

144

11

12

2.1

19

4.5

30

8.3

13

5.3

+

150

154

14.6

14

1.7

23

1.0

27

3.6

13

4.2

+

500

38

5.3

5

4.0

27

6.1

17

7.2

4

1.7

+

1500

0V

0.0

0V

0.0

20

5.5

0V

0.0

0V

0.0

+

5000

N/T

N/T

0V

0.0

N/T

N/T

Positive controls

S9-Mix

+

 

 

 

Name

Concentration µg/plate)

Nº colonies per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

2122

505.5

212

41.0

479

7.6

231

25.4

546

60.5

 BP        Benzo(a)pyrene                                        N/T   Not testedat this dose level

2AA     2-Aminoanthracene                                      S      Sparse bacterial background lawn

V          Very weak bacterial background lawn           #      Standard deviation

*           p¿ 0,05

 

 Experiment 2

 

Table 3: without metabolic activation

With or without S9-Mix

Test substance concentration (µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

 

 TA100           TA1535     WP2uvrA

Frameshift type

    

  TA98           TA1537

-

0

81

12.3#

19

1.7

18

6.1

15

2.3

9

5.3

-

5

89

1.0

16

3.2

N/T

14

2.1

N/T

-

15

79

15.0

15

6.2

N/T

14

1.5

9

1.2

-

50

85

3.2

22

7.0

20

9.8

16

2.3

10

3.8

-

100

N/T

N/T

N/T

N/T

13

3.2

-

150

76

9.6

15

1.2

17

3.8

15

3.1

9

4.4

-

200

N/T

N/T

N/T

N/T

6

1.5

-

500

10S

6.0

3S

1.0

16

3.8

6S

3.1

0V

0.0

-

1500

0V

0.0

0V

0.0

14

3.6

0V

0.0

N/T

-

5000

N/T

N/T

2

1.7

N/T

N/T

Positive controls

S9-Mix

-

 

 

Name

 

ENNG

ENNG

ENNG

4NQO

9AA

Concentration

(µg/plate)

3

5

2

0.2

80

No. colonies per plate

 

359

12.2

269

35.5

817

40.6

89

8.4

940

164.2

 

ENNG   N-ethyl-N'-nitro-N-nitrosoguanidine                  N/T   Not tested at this dose level

4NQO   4-Nttroquinoline-l-oxide                                   S      Sparse bacterial background lawn

9AA      9-Aminoacridine                                            V      Very weak bacterial background lawn

#          Standard deviation

 

Table 4: with metabolic activation

With or without

S9-Mix

 

Test substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

  TA100        TA1535       WP2uvrA

Frameshift type

TA98        TA1537

+

0

93

4.0#

12

4.5

21

2.6

22

3.1

10

4.2

+

5

97

8.2

13

2.3

N/T

23

4.6

N/T

+

15

83

11.8

12

1.7

N/T

17

3.5

11

3.1

+

50

91

12.1

12

1.0

20

2.5

29

9.2

10

2.0

 

100

N/T

N/T

N/T

N/T

13

1.7

+

150

82

12.7

13

2.1

20

3.5

31

12.4

17

6.6

+

200

N/T

N/T

N/T

N/T

11

4.6 

+

500

30

10.8

4S

1.7

19

5.3

11S

4.2

5S

2.5

+

1500

0V

0.0

0V

0.0

13

3.1

0V

0.0

N/T

+

5000

N/T

N/T

2S

2.1

N/T

N/T

Positive controls

S9-Mix

+

 

 

Name

 

2AA

2AA

2AA

BP

2AA

Concentration (µg/plate)

1

2

10

5

2

No. colonies per plate

929

68.6

128

36.0

708

24.3

174

36.3

435

56.5

 

BP       Benzo(a)pyrene                      N/T   Not tested at this dose level

2AA     2-Aminoanthracene                  S      Sparse bacterial background lawn

#         Standard deviation                    V      Very weak bacterial background lawn

 

 

Experiment 3

Table 5: with and without metabolic activation

Test substance concentration µg/plate)

Number of revertants (mean number of colonies per plate)

Frameshift type

(Without metabolic activation)

TA1537

Frameshift type

(With metabolic activation)

TA1537

0

8

3.2#

12

3.1

15

9

2.0

11

4.2

50

10

1.5

13

2.6

100

9

0.7

13

4.5

150

11

3.2

13

3.5

200

2S

1.7

14

4.0

500

0V

0.0

10S

7.8

Name

9AA

2AA

Concentration (µg/plate)

80

2

No. colonies per plate

940

87.0

399

137.2

9AA     9-Aminoacridine

2AA     2-Arninoanthracene

S         Sparse bacterial background lawn

V         Very weak bacterial background lawn

#          Standard deviation

Conclusions:
The test material was considered to be non-mutagenic under the conditions of this test and therefore is not to be classified according to CLP Regulation.
Executive summary:

The Bacterial Reverse mutation Assay (Ames test) for the test substance was performed with five histidine dependents bacteria, four strains of Salmonella typhimurium T1535, TA98, TA100, and TA1537 and E.Coli WP2 uvrA. Plate Incorporation Method was carried out at the concentrations of 5000, 1500, 500, 150, 50 and 15 µg/plate for the strains of Salmonella typhimurium in the presence and absence of metabolic activation and at the concentrations of 1500, 500, 150, 50, 15 and 5 µg/plate for the E.coli WP2 uvrA in the presence and absence of metabolic activation.The dose range for TA1537 (with/without S9) was modified slightly, based on results observed in the Experiment 1 and was 500, 200, 100, 50 and 15 µg/plate. Intermediate doses were included to allow for small but statistically significant increases observed in the first experiment. The test material caused a visible reduction in the growth of the bacterial lawn to all of the bacterial tester strains both with and without metabolic activation. The toxicity of the test material to the bacterial tester strains varied both between strains and between exposures with or without S9-mix. The test material was, therefore, tested up to its toxic limit. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Very small, statistically significant increases were observed to TA1537 in Experiment 1 only both with and without S9. However, these increases were not considered to be biologically significant as they were within the strains historical range and were non-reproducible over three separate experiments. According to the obtained results, the test substance was, therefore, considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 26, 2000 to September 7, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability.
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Preliminary Toxicity test: 0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/ml
Experiment 1: with and without S9: 0, 9.77, 19.53, 39.06, 78.13, 156.25, 234.38 and 312.5 µg/ml
Experiment 2: without S9: 0, 4.89, 9.77, 19.53, 39.06, 78.13, 117.19 µg/ml
Experiment 2: with S9: 0, 19.53, 39.06, 78.13, 156.25, 234.38 and 312.5 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Eagle's minimal essential medium (MEM)
Negative solvent / vehicle controls:
yes
Remarks:
Eagle's minimal essential medium (MEM)
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without metabolic activation system
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation system
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium. 1.0 ml of the appropriate solution of vehicle, positive control or test material was added to 9.0 ml 0f each culture. 1 ml of 10% S9 (ie 1 % final concentration of S9) was used for the Preliminary Toxicity Test and Experiment 1, and 1 ml of 20% S9 (ie 2% final concentration of S9) for Experiment 2.
- Exposure duration and Expression time (cells in growth medium):
Experiment 1: with S9-mix was exposed 4 hours with a cell harvest after a 20-hour expression period.
Experiment 1: without S9-mix was exposed 4 hours with a cell harvest after a 20-hour expression period.
Experiment 2: with S9-mix was exposed 4 hours with a cell harvest after a 20-hour expression period.
Experiment 2: without S9-mix the exposure time was 24 hours.
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours, the mitosis was arrested two hours before the required harvest time

SELECTION AGENT (mutation assays): Colcemid
STAIN (for cytogenetic assays): the slides were stained in 5% Gurrs Giemsa for 5 minutes.

NUMBER OF REPLICATIONS: all cultures were set up in duplicate.

NUMBER OF CELLS EVALUATED: where possible the first 100 consecutive well-spread metaphases from each culture were counted

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index. A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
If the metaphase cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides. A statistically significant increase in the frequency of cells with chromosome aberrations (excluding gaps) is considered as a positive result.
Statistics:
The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In all cases test substance showed some toxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: there was no observable change in pH when the test substance was dosed into the media.
- Effects of osmolality: the osmolality did not increase (50 mOSM)
- Precipitation: there was no precipitate of the test material seen in the cultures.

COMPARISON WITH HISTORICAL CONTROL DATA: The vehicle control cultures had frequencies of cell with chromosome aberrations within the expected range and the positive control treatments gave statistically significant increases in the frequency of cells with aberrations.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Metaphase cells were present at dose levels up to 2500 µg/ml in the 4(20) hour treatment in the absence of metabolic activation (S9) and up to 156.25 µg/ml in the 4(20) hour treatment in the presence of S9. The maximum dose with metaphases present in the 24-hour continuous exposure was 78.13 µg/ml. In the 4 hour exposure group without S9, approximately 50% mitotic inhibition was achieved at 156.25 µg/ml and complete mitotic inhibition at 5000 µg/ml.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1:Mitotic Index Experiment 1

MITOTIC INDEX - EXPERIMENT 1 (4-HOUR TREATMENT, 24-HOUR HARVEST)

DOSE LEVEL

4 HOURS TREATMENT WITHOUT S9

4 HOURS TREATMENT WITH S9

µg/ml

A

B

Mean

% of

Control

A

B

Mean

% of

Control

0

4.20

6.20

5.20

100

6.05

6.15

6.10

100

9.77

-

-

-

-

-

-

-

-

-

19.53

-

-

-

-

-

-

-

-

-

39.06

3.80

5.70

4.75

91

7.05

6.95

6.95

7.00

-

78.1 3

4.65

4.55

4.60

88

6.40

5.60

5.60

6.00

98

156.25

2.65

1.50

2.08

40

J.40

2.25

2.25

2.83

46

234.38

NM

NM

0.00

0

NM

NM

NM

0.00

0

312.5

NM

NM

0.00

0

NM

NM

NM

0.00

0

MMC 0.4

2.75

2.20

2.48

48

NA

NA

NA

NA

NA

CP 25

NA

NA

NA

NA

1.95

1.05

1.95

1.50

25

MMC: mitomycin C              CP: cyclophosphamide        - : not assessed for mitotic index            NA: not applicable

NM: no scorable metaphases

 

Table 2: Mitotic Index Experiment 2 

MITOTIC INDEX - EXPERIMENT 2 (24-HOUR HARVEST)

DOSE LEVEL

24 HOURS TREATMENT WITHOUT S9

4 HOURS TREATMENT WITH S9

µg/ml

A

B

Mean

% of

Control

A

B

Mean

% of

Control

0

4.35

3.65

4.00

100

3.90

4.50

4.20

100

4.89

-

-

-

-

NA

NA

NA

NA

9.77

-

-

-

-

NA

NA

NA

NA

19.53

-

-

-

-

-

-

-

-

39.06

4.35

2.90

3.63

91

5.00

4.55

4.78

1 14

78.13

2.55

2.60

2.58

65

6.45

4.00

5.23

125

11 7.19

1.10

1.40

1.25

31

NA

NA

NA

NA

156.25

NA

NA

NA

NA

1.95

1.30

1.63

39

234.38

NA

NA

NA

NA

NM

NM

0.00

0

312.5

NA

NA

NA

NA

NM

NM

0.00

0

MMC 0.2

1.50

0.95

1.23

31

NA

NA

NA

NA

CP 25

NA

NA

NA

NA

0.60

0.35

0.48

11

 MMC: mitomycin C              CP: cyclophosphamide        - : not assessed for mitotic index            NA: not applicable

NM: no scorable metaphases

Conclusions:
The test substance was shown to be non-clastogenic to human lymphocytes in vitro and therefore is not to be classified according to CLP Regulation.
Executive summary:

The In Vitro Mammalian Chromosome Aberration Test for the test substance was performed with human lymphocytes. A total of two experiments with four treatment conditions were used for the study, ie. 4 hours exposure with the addition of metabolic activation system (S9) with cell harvest after a 20-hour expression period and a 4-hour exposure in the absence of activation with a 20-hour expression period, this was Experiment 1. In the Experiment 2 the 4-hour exposure with addition of S9 was repeated (using a 2% final concentration of S9), whilst in the absence of activation the exposure time was increased to 24 hours. For the metaphase analysis the dose levels selected were: 39.06, 78.13, and 156.25 µg/ml in the Experiment 1; 39.06, 78.13 and 117.19 µg/ml in the Experiment 2, treatment time 24 hours, and 39.06, 78.13 and 156.25 µg/ml in the Experiment 2, treatment time 4 hours. The test substance showed some evidence of toxicity in all cases, a complete mitotic inhibition was observed in the 4 hour exposure group without S9, at 5000 µg/ml. The test substance did not induce a statistically significant increase in the frequency of cells with chromosome aberrations (excluding gaps) in either the presence or absence of a metabolic activation system in either of two separate experiments. The test substance was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 11, 2009 to September 28, 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase, TK +/-
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Experiment 1, without metabolic activation: 5, 10, 20, 30, 40, 45, 50 and 55 µg/ml
Experiment 1, with metabolic activation: 20, 30, 40, 50, 55, 60, 65 and 70 µg/ml
Experiment 2, without metabolic activation: 1.25, 2.5, 5, 7.5, 10, 15, 20 and 25 µg/ml
Experiment 2, with metabolic activation: 10, 20, 30, 40, 50, 55, 60 and 65 µg/ml
Vehicle / solvent:
R0 medium
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation; 400 and 150 µg/mL ethylmethanesulphonate for Experiment 1 and Experiment 2, respectively
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation; cyclophosphamide 2 µg/ml
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment 1: several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10E06 cells/ml in 10 ml aliquots in R10 medium in sterile plastic universals. To each universal was added 2 ml of S9-mix if required, 2 ml of the treatment dilutions and sufficient R0 medium to bring the total volume to 20 ml. The treatment vessels were incubated at 37 ºC for 4 hours with continuous shaking using an orbital shaker within an incubated hood.
Experiment 2: As in Experiment 1, an exponentially growing stock culture of cells was established. The cells were counted and processed to give 1 x 10E06 cells/ml in 10 duplicate cultures in R10 medium for the 4-hour treatment with metabolic activation cultures. In the absence of metabolic activation the exposure period was extended to 24 hours therefore 0.3 x 10E06 cells/ml in 10 ml duplicate cultures were established in 25 cm2 tissue culture flasks. To each culture 2 ml of S9-mix was added if required, 2.0 ml of the treatment dilutions and sufficient R0 medium to give a final volume of 20 ml. The treatment vessels were incubated at 37 ºC with continuous shaking using an orbital shaker for 24 hours in the absence of S9-mix and 4 hours in the presence of S9-mix.

DURATION
- Exposure duration:
Experiment 1: 4 hours
Experiment 2, without metabolic activation: 24 hours
Experiment 2, with metabolic activation: 4 hours
- Expression time (cells in growth medium): two days.

SELECTION AGENT (mutation assays): On day 2 of the experiment, the cells were counted, diluted to 10E04 cells/ml and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/ml 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/ml and plated (2 cells/well) for viability (%V) in non-selective medium. The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

NUMBER OF REPLICATIONS: Duplicates

DETERMINATION OF CYTOTOXICITY
A preliminary toxicity test was performed on cell cultures at 5 x 10E05 cells/ml, using a 4-hour exposure time both with and without metabolic activation (S9) and at 1.5 x 10E05 cells/ml using a 24-hour exposure without S9. The dose range used in the preliminary toxicity test was 19.53 to 5000 µg/ml for all three of the exposure groups initially. However, due to the very high levels of toxicity observed, a repeat of the test was performed using a dose range of 5 to 80 µg/ml for the 4-hour exposure groups and 0.63 to 50 µg/ml for the 24-hour exposure group. Following the exposure period, the cells were washed twice with R10, resuspended in R20 medium, counted using a coulter counter and then serially diluted to 2 x 10E05 cells/ml. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth values (SG). The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a % Relative Suspension Growth Value (%RSG).





METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period:
- Exposure duration:
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS:

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:
Evaluation criteria:
For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. Following discussions at an International Workshop on Genotoxicity Test Procedures in Plymouth, UK, 2002 (Moore at al 2003) it was felt that the IMF must exceed some value based on the global background MF for each method (agar or microwell). This Global Evaluation Factor (GEF) value was set following a further meeting of the International Workshop in Aberdeen, Scotland, 2003 (Moore et al 2006) at 126 x 10-6 for the microwell method. Therefore, any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10-6 will be considered positive. However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test material induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
Statistics:
The experimental data was analysed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1: at 55 µg/ml in the absence of metabolic activation, and at and above 60 µg/ml in the presence of metabolic activation; Experiment 2: at 20 µg/ml in the absence of metabolic activation and at 55 µg/ml in the presence of metabolic activation
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Experiment 1: there was evidence of toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values. There was no evidence of any significant reductions in (%V) viability, therefore, indicating that no residual toxicity had occurred in either of the exposure groups. Optimum levels of toxicity were achieved in both the absence and presence of metabolic activation. The excessive toxicity observed at 55 µg/ml in the absence of metabolic activation, and at and above 60 µg/ml in the presence of metabolic activation, resulted in these doses not being plated for viability or 5-TFT resistance. The toxicity observed at 55 µg/ml in the presence of metabolic activation exceeded the upper acceptable limit of 90%, therefore, this dose was excluded from the statistical analysis. Acceptable levels of toxicity were seen with both positive control substances.

As was seen in Experiment 1 and the preliminary toxicity test there was evidence of dose related reductions in % RSG and RTG values in cultures dosed with the test material in both the absence and presence of metabolic activation. There was no evidence of any significant reductions in (%V) viability, therefore, indicating that no residual toxicity had occurred in either of the exposure groups. Optimum levels of test material-induced toxicity were achieved in both the absence and presence of metabolic activation. The excessive toxicity observed at 25 µg/ml in the absence of metabolic activation and at and above 60 µg/ml in the presence of metabolic activation, resulted in these doses not being plated for viability or 5-TFT resistance. The toxicity observed at 20 µg/ml in the absence of metabolic activation and at 55 µg/ml in the presence of metabolic activation, exceeded the upper acceptable limit of 90%, therefore, these doses were excluded from the statistical analysis. The 24-hour exposure without metabolic activation treatment, demonstrated that the extended time point had a marked effect on the toxicity of the test material.

The test material did not induce any statistically significant or dose related increases in the mutant frequency x 10-6 per viable cell in the presence of metabolic activation. In the absence of metabolic activation, a small but statistically significant increase in mutant frequency was observed at the upper surviving dose level. However, the increase in mutant frequency observed at this dose level did not exceed the GEF value and the mutant frequency value was within the acceptable range for vehicle controls. Therefore, the response was considered to be of no toxicological significance. Precipitate of test material was not observed at any of the dose levels.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Summary of results

 

Experiment 1

 

Treatment

(µg/ml)

4-hours-S9

Treatment

(µg/ml)

4-hours+S9

%RSG

RTG

MF§

%RSG

RTG

MF§

0

100

1.00

128.81

0

100

1.00

110.66

 

96

 

 

20

104

1.04

133.82

10

104

1.07

127.02

30

94

1.13

93.18

20

91

0.92

114.98

40

73

0.85

75.02

30

89

0.87

101.25

50

18

0.17

112.00

40

64

0.67

114.96

55x

2

0.04

93.96

45

43

0.41

111.80

60ø

0

 

 

50

17

0.14

100.71

65ø

0

 

 

55ø

1

 

 

70ø

0

 

 

Linear trend NS

Linear trend NS

EMS

 

 

 

CP

 

 

 

400

69

0.35

1075.82

2

48

0.16

1083.23

 

Experiment 2

 

Treatment

(µg/ml)

24-hours-S9

Treatment

(µg/ml)

4-hours+S9

%RSG

RTG

MF§

%RSG

RTG

MF§

0

100

1.00

101.57

0

100

1.00

86.58

ø

94

 

 

10

103

0.91

131.57

2.5

91

0.96

112.32

20

101

1.23

99.26

5

72

0.77

112.32

30

85

0.98

130.53

7.5

57

0.58

95.39

40

72

0.80

133.19

10

44

0.44

120.11

50

21

0.22

69.10

15

23

0.16

180.49*

55x

0

0.02

90.24

20x

6

0.07

164.09

60 ø

0

 

 

25 ø

1

 

 

65 ø

0

 

 

Linear trend *

Linear trend NS

EMS

 

 

 

CP

 

 

 

150

63

0.45

1036.81

2

50

0.26

750.46

 

Conclusions:
The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test. It is not classified according to the CLP Regulation.
Executive summary:

The study was conducted according to a method that was designated to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD guideline 476 and EU method B.17. Two experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2.c mouse lymphoma cells were treated with the test material at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4 -hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at eight dose levels using a 4 -hour exposure group in the presence of metabolic activation (1% S9) and a 24 -hour exposure group in the absence of metabolic activation. The dose range of the test material was selected following the results of a preliminary toxicity test. The dose range for Experiment 1 was 5 to 55 µg/ml in the absence of metabolic activation and 20 to 70 µg/ml in the presence of metabolic activation. The dose range for Experiment 2 was 1.25 to 25 µg/ml in the absence of metabolic activation and 10 to 65 µg/ml in the presence of metabolic activation. The maximum dose level used was limited by test material induced toxicity. Precipitate of test material was not observed at any of the dose levels. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first of the second experiment and at dose levels which achieved optimum levels of toxicity. The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Bacterial reverse mutation assay: Key study: Test according to OECD guideline 471 and EU guideline B14. GLP study. The substance was considered to be non-mutagenic.

Chromosomal aberrations in mammalian cells: Key study: Test according to OECD guideline 473 and EU guideline B.10. GLP study.

The substance was shown to be non-clastogenic to human lymphocytes in vitro.

Gene mutation in mammalian cells: Key study: Test according to OECD guideline 476 and EU guideline B.17. GLP study.

The substance did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic.

Short description of key information:

Bacterial reverse mutation assay: Key study: Test according to OECD guideline 471 and EU guideline B14. GLP study. The substance was considered to be non-mutagenic.

Chromosomal aberrations in mammalian cells: Key study: Test according to OECD guideline 473 and EU guideline B.10. GLP study.  The substance was shown to be non-clastogenic to human lymphocytes in vitro.

Gene mutation in mammalian cells: Key study: Test according to OECD guideline 476 and EU guideline B.17. GLP study.

The substance did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available information on genetic toxicity in vitro, the substance is considered to be negative and therefore, the substance is not classified.