Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
The genotoxic potential of the test substance, TM 10-202, was assessed as negative in the presence and absence of metabolic activation according to OECD Test Guideline 471 using the Bacterial Reverse Mutation method.
Link to relevant study records
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:
The study was conducted between 25 June 2010 and 22 July 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 471 using the Bacterial Reverse Mutation method and in compliance with GLP.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
Salmonella typhimurium: Histidine
Escherichia coli: Tryptophan
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.

Mutation Test – Experiment 1 (Range-finding Test):
Salmonella strains (without S9-mix): 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate.
Salmonella strains (with S9-mix): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
WP2uvrA- (with and without S9-mix): 50, 150, 500, 1500 and 5000 µg/plate.

Mutation Test – Experiment 2 (Main Test)
Salmonella strain TA100 (without S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 µg/plate.
Salmonella strains TA1535 and TA1537 (without S9-mix): 0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate.
Salmonella strain TA98 (without S9-mix): 0.015, 0.05, 0.15, 0.5, 5 and 50 µg/plate.
All Salmonella strains (with S9-mix): 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate.
WP2uvrA- (with and without S9-mix): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Vehicle:
In solubility checks performed in house, the test item was noted to be immiscible in sterile distilled water and only partially miscible in dimethyl sulphoxide at 50 mg/ml. However, the test item was fully miscible in acetone at the same concentration (50 mg/ml) therefore, acetone was selected as the vehicle.
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and conditions:
Tester strains
The four strains of Salmonella used in the test were obtained either from the University of California, Berkeley, on culture discs, on 04 August 1995 or from Syngenta CTL, Alderley Edge, as frozen vials, on 20 March 2007. E. coli strain WP2uvrA- was obtained from the British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987. All of the strains were stored at approximately ¯196°C in a Statebourne liquid nitrogen freezer, model SXR 34. Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate (Mortelmans and Zeiger (2000)).
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37ºC for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Preparation of Test Item
The test item was accurately weighed and appropriate dilutions prepared in acetone by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (6.8%) of the test item. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino silicate pellets with a nominal pore diameter of 4 x E-04 microns. All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Preliminary toxicity test
In order to select appropriate dose levels for use in the main test, a preliminary assay was carried out to determine the toxicity of the test item. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The assay was performed by mixing 0.1 ml of bacterial culture (TA100 or WP2uvrA-), 0.1 ml of test item formulation, 0.5 ml of S9-mix or phosphate buffer and 2 ml of molten, trace histidine or tryptophan supplemented, top agar and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Ten concentrations of the test item and a vehicle control (acetone) were tested. In addition, 0.1 ml of the maximum concentration of the test item and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test item. After approximately 48 hours incubation at 37ºC the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Mutation test - Experiment 1 (Range-Finding test).
Up to seven concentrations of the test item (ranging between 0.5 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method. The dose ranges were allocated as follows:
Salmonella strains (without S9-mix): 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate.
Salmonella strains (with S9-mix): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
WP2uvrA- (with and without S9-mix): 50, 150, 500, 1500 and 5000 µg/plate.
Additional dose levels and expanded dose range were selected (where applicable) in order to achieve both four non-toxic doses and the toxic limit of the test item.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test item formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix.
All of the plates were incubated at 37ºC for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

Mutation test - Experiment 2 (Main test).
The second experiment was performed using fresh bacterial cultures, test item and control solutions. The test item dose range was amended following the results of the range-finding test and ranged between 0.015 and 5000 µg/plate. The dose ranges were allocated as follows:
Salmonella strain TA100 (without S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 µg/plate.
Salmonella strains TA1535 and TA1537 (without S9-mix): 0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate.
Salmonella strain TA98 (without S9-mix): 0.015, 0.05, 0.15, 0.5, 5 and 50 µg/plate.
All Salmonella strains (with S9-mix): 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate.
WP2uvrA- (with and without S9-mix): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Additional dose levels and expanded dose range were again selected in order to achieve both four non-toxic doses and the toxic limit of the test item.
The test item formulations and vehicle control were dosed using the pre-incubation method as follows:
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 ml of S9-mix or phosphate buffer and 0.05 ml of the vehicle or test item formulation and incubated for 20 minutes at 37°C with shaking at approximately 130 rpm prior to the addition of 2 ml of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix.
All of the standard plates were incubated at 37ºC for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.
Evaluation criteria:
The reverse mutation assay may be considered valid if the following criteria are met:

Tester Strain Integrity
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Although the number of spontaneous revertants can be expected to fall within the ranges, they may occasionally fall outside these. The experiment may, however, still be considered valid if the organisms respond normally to their respective positive controls.

Tester strain Culture Density
To demonstrate that appropriate numbers of bacterial cells have been plated, the bacterial cell count for each tester strain culture should be in the range of 0.9 to 9 E+09 bacteria per ml.

Positive Control Values
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study must induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.

Cytotoxicity
Generally, a minimum of four non-toxic test item dose levels is required for the assay to be acceptable.

Contamination
There should be no evidence of excessive contamination. However, if a small number of plates are affected by sporadic airborne contaminants then the experiment will be assessed and accepted provided that the integrity of the study is not compromised and that the contamination is not systematic.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
other: A toxic response was noted for the Salmonella strains with and without S9-mix. No toxicity was observed to E.coli strain WP2uvrA- in the presence of S9 although weakened lawns were noted in the absence of S9-mix.
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
Preliminary toxicity test
The test item was toxic at and above 50 µg/plate to TA100 and non toxic to WP2uvrA-. The test item formulation and S9-mix used in this experiment were both shown to be sterile.

Mutation test
Prior to use, the properties of the S typhimurium strains and the E coli strain regarding the membrane permeability, ampicillin resistance, biotin/tryptophan dependence, normal spontaneous mutation rates and viability was performed as defined by Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

In the range-finding test (plate incorporation), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains initially from 50 and 500 µg/plate in the absence and presence of S9-mix, respectively. No toxicity was noted to Escherichia coli strain WP2uvrA- at any dose level either in the absence or presence of S9-mix. In the main test (pre-incubation), a stronger test item induced toxic response was noted with visible reductions in the growth of the bacterial background lawns noted for the Salmonella strains from 5 and 150 µg/plate in the absence and presence of S9-mix, respectively. No toxicity was observed to E.coli strain WP2uvrA- in the presence of S9 although weakened lawns were noted in the absence of S9-mix from 500 µg/plate. The sensitivity of the bacterial tester strains to the toxicity of the test item varied between bacterial strain type, exposures with and without S9-mix and methodology. The test item was, therefore, either tested up to the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial strain type, exposure to S9 mix and experiment number. An oily, globular precipitate was observed at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Preliminary toxicity test

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

143

116

131

134

97

105

90 *

108 *

33 *

8 *

11*P

+

TA100

88

87

73

100

100

69

81

94

29 *

15 *

8 *P

-

WP2uvrA-

34

21

24

20

24

36

35

20

25

20

27 P

+

WP2uvrA-

33

20

26

26

30

28

26

38

20

29

32 P

* Partial absence of bacterial background lawn

P Precipitate

Spontaneous Mutation Rates (Concurrent Negative Controls)

Range-finding Test

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

98

 

20

 

35

 

27

 

12

 

100

(99)

21

(20)

51

(44)

22

(24)

14

(13)

98

 

18

 

46

 

22

 

13

 

Main Test

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

93

 

13

 

47

 

22

 

10

 

99

(93)

18

(16)

55

(47) ††

15

(18)

7

(9)

86

 

18

 

39

 

17

 

9

 

102

 

 

31

 

 

97

(96) †

15

(25) †

89

 

28

 

         Experintal procedure perford at a later date (without S9-mix only) due to excessive toxicity in the original test

††       Experintal procedure perford at a later date (with and without S9-mix) due to elevated solvent control values in the original test

 

Test Results: Range-Finding Test– Without Metabolic Activation

Test Period

From: 01 July 2010

To: 04 July 2010

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

-

0

101

106

101

(103)

2.9#

20

18

13

(17)

3.6

43

42

51

(45)

4.9

15

26

22

(21)

5.6

7

13

13

(11)

3.5

-

0.5

107

98

107

(104)

5.2

12

24

23

(20)

6.7

N/T

25

18

25

(23)

4.0

12

11

12

(12)

0.6

-

1.5

103

101

98

(101)

2.5

23

25

21

(23)

2.0

N/T

22

25

31

(26)

4.6

13

5

14

(11)

4.9

-

5

107

92

95

(98)

7.9

20

30

16

(22)

7.2

N/T

23

26

18

(22)

4.0

15

14

11

(13)

2.1

-

15

99

108

108

(105)

5.2

18

12

16

(15)

3.1

N/T

20

21

11

(17)

5.5

10

12

8

(10)

2.0

-

50

106 *

100 *

113 *

(106)

6.5

13

13

8

(11)

2.9

46

41

51

(46)

5.0

24

12

22

(19)

6.4

10

11

12

(11)

1.0

-

150

111 *

96 *

99 *

(102)

7.9

19 *

12 *

14 *

(15)

3.6

44

40

47

(44)

3.5

10 *

15 *

10 *

(12)

2.9

7 *

10 *

10 *

(9)

1.7

-

500

84 *

67 *

78 *

(76)

8.6

16 *

14 *

21 *

(17)

3.6

48

47

34

(43)

7.8

11 *

14 *

9 *

(11)

2.5

5 *

10 *

7 *

(7)

2.5

-

1500

N/T

N/T

41

27

42

(37)

8.4

N/T

N/T

-

5000

N/T

N/T

26 P

30 P

43 P

(33)

8.9

N/T

N/T

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

515

521

441

(492)

44.6

145

146

145

(145)

0.6

263

258

214

(245)

27.0

143

128

120

(130)

11.7

2614

1283

1645

(1847)

688.2

 ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

N/T     Not tested at this dose level

P        Precipitate

*         Partial absence of bacterial background lawn

#        Standard deviation

 

Test Results: Range-Finding Test– With Metabolic Activation

Test Period

From: 01 July 2010

To: 04 July 2010

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

+

0

92

79

104

(92)

12.5#

11

15

9

(12)

3.1

60

42

53

(52)

9.1

29

24

29

(27)

2.9

15

13

18

(15)

2.5

+

5

70

73

100

(81)

16.5

13

16

16

(15)

1.7

N/T

27

30

30

(29)

1.7

10

12

15

(12)

2.5

+

15

86

97

54

(79)

22.3

10

18

16

(15)

4.2

N/T

34

16

30

(27)

9.5

10

14

14

(13)

2.3

+

50

81

82

90

(84)

4.9

18

12

15

(15)

3.0

53

56

55

(55)

1.5

27

29

18

(25)

5.9

13

13

12

(13)

0.6

+

150

73

85

82

(80)

6.2

14

5

18

(12)

6.7

56

47

52

(52)

4.5

24

27

18

(23)

4.6

7

12

11

(10)

2.6

+

500

85 *

67 *

86 *

(79)

10.7

12

16

14

(14)

2.0

52

38

53

(48)

8.4

26 *

13 *

21 *

(20)

6.6

5

5

10

(7)

2.9

+

1500

34 *

53 *

52 *

(46)

10.7

13 *

11 *

10 *

(11)

1.5

42

44

37

(41)

3.6

9 *

14 *

4 *

(9)

5.0

4 *

1 *

9 *

(5)

4.0

+

5000

35 P *

30 P *

34 P *

(33)

2.6

13 P *

13 P *

14 P *

(13)

0.6

37 P

40 P

41 P

(39)

2.1

7 P *

0 P *

4 P *

(4)

3.5

2 P *

1 P *

2 P *

(2)

0.6

Positive

controls

 

S9-Mix

 

+

Name

Concentration

(μg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

1530

1286

1379

(1398)

123.1

187

144

159

(163)

21.8

256

274

239

(256)

17.5

200

185

177

(187)

11.7

167

143

142

(151)

14.2

BP      Benzo(a)pyrene

2AA    2-Aminoanthracene

N/T     Not tested at this dose level

P        Precipitate

*         Partial absence of bacterial background lawn

#         Standard deviation

 

Conclusions:
Interpretation of results (migrated information):
negative

The test item, TM 10-202, was considered to be non-mutagenic under the conditions of this test.
Executive summary:

The genotoxic potential of the test substance, TM 10-202, was assessed as negative in the presence and absence of metabolic activation according to OECD Test Guideline 471 using the Bacterial Reverse Mutation method.

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

Additional information

Additional information from genetic toxicity in vitro:

Justification for selection of genetic toxicity endpoint
The study was conducted on the target substance in appropriate test species and according to internationally recognised guidelines.

Justification for classification or non-classification

This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.

To arrive at a classification, test results are considered from experiments determining mutagenic and genotoxic effects in germ and/or somatic cells of exposed animals and in in vitro tests.

The system is hazard based, classifying substances on the basis of their intrinsic ability to induce mutations in germs cells, and does not give a quantitative assessment of the risk.

To this end, the test substance has been assessed according to internationally recognized guidelines in an in vitro gene mutation study in bacteria (Ames test). 

 

In the Ames test, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains tested with any dose of the test item, either with or without metabolic activation.

 

Based on the negative results in vitro, the test item is considered non-mutagenic.