Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative with S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538 and E. coli WP2 uvrA with and without metabolic activation

Ames test (OECD 471): negative with S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538 with and without metabolic activation

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:
19 Aug - 23 Sep 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon (for S. typhimurium strains)
trp operon (for E. coli strain)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
Test concentrations with justification for top dose:
Pre-experiment: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation.

The pre-experiment is reported as Experiment I.

Experiment II: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to bacteria.
Untreated negative controls:
yes
Remarks:
untreated controls
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (Experiment I); preincubation (Experiment II)

DURATION
- Preincubation period: 1 h
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiment

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn
Evaluation criteria:
A test item is considered as a mutagenic if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose-dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose-dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
Mean values and standard deviations were calculated.
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:
cytotoxicity
Remarks:
in strains TA 1537, TA 98 and TA 100 with and without S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
Remarks:
but tested up to the precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated in the overlay agar in the test tubes at 5000 µg/plate with and without S9 mix and in Experiment II from 2500 to 5000 µg/plate with S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in Experiment I at 5000 µg/plate. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment is reported as Experiment I (all strains were tested in the pre-experiment).

ADDITIONAL INFORMATION ON CYTOTOXICITY: The plates incubated with the test item showed reduced background growth in strains TA 1537, TA 98 and TA 100 with and without metabolic activation. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in strain TA 1537 with metabolic activation (experiment 2) and in strains TA 98 and TA 100 with and without metabolic activation (experiment 2 and experiment 1 and 2. respectively). Please refer to table 3 and 4.

Table 1. Test results of main test 1 (plate incorporation).

With or without S9-Mix

Test substance concentration [μg/plate]

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA1535

TA100

WP2 uvrA

TA1537

TA98

-

0 (DMSO)

11 ± 3

154 ± 16

35 ± 1

9 ± 1

26 ± 6

-

0

13 ± 3

172 ± 9

38 ± 5

14 ± 0

23 ± 4

-

3

12 ± 2

160 ± 23

38 ± 6

9 ± 2

26 ± 4

-

10

12 ± 3

167 ± 16

43 ± 3

8 ± 3

25 ± 2

-

33

10 ± 2

161 ± 8

41 ± 10

8 ± 2

25 ± 3

-

100

9 ± 1

138 ± 24

38 ± 3

9 ± 1

21 ± 7

-

333

9 ± 1

56 ± 7R

39 ± 10

9 ± 3R

31 ± 2R

-

1000

8 ± 2

55 ± 4R

37 ± 9

6 ± 2R

27 ± 4R

-

2500

7 ± 2

54 ± 12R

37 ± 6

5 ± 2R

25 ± 5R

-

5000

10 ± 4P

63 ± 8P,R

33 ± 4P

5 ± 2P,R

20 ± 5P,R

Positive controls, –S9

Name

NaN3

NaN3

MMS

4-NOPD

4-NOPD

Concentration

[μg/plate]

10

10

2.0 µL

50

10

Mean No. of colonies/plate

(average of 3 ± SD)

1136 ± 68

2125 ± 126

829 ± 40

78 ± 6

336 ± 30

+

0 (DMSO)

11 ± 3

141 ± 7

48 ± 5

14 ± 2

30 ± 3

+

0

11 ± 4

170 ± 2

56 ± 11

13 ± 1

27 ± 4

+

3

11 ± 1

142 ± 11

49 ± 7

13 ± 2

30 ± 3

+

10

11 ± 4

171 ± 19

47 ± 5

11 ± 3

32 ± 2

+

33

10 ± 5

139 ± 14

43 ± 3

11 ± 2

37 ± 9

+

100

9 ± 3

150 ± 17

50 ± 5

13 ± 2

36 ± 2

+

333

7 ± 1

119 ± 24

46 ± 8

15 ± 1

32 ± 3

+

1000

9 ± 3

38 ± 3R

43 ± 8

12 ± 1M,R

29 ± 6R

+

2500

10 ± 4

33 ± 2M,R

46 ± 2

13 ± 1M,R

23 ± 2R

+

5000

12 ± 2P

29 ± 2P,M,R

37 ± 6P

13 ± 2P,M,R

16 ± 2P,M,R

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentration

[μg/plate]

2.5

2.5

10

2.5

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

348 ± 19

3985 ± 50

384 ± 50

197 ± 5

4320 ± 413

NaN3: sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

M: manual count

P: precipitate

R: reduced background growth

 

Table 2. Test results of main test 2 (preincubation).

With or without S9-Mix

Test substance concentration

g/plate]

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA1535

TA100

WP2 uvrA

TA1537

TA98

-

0 (DMSO)

12 ± 3

143 ± 9

36 ± 6

8 ± 2

30 ± 10

-

0

10 ± 3

201 ± 2

39 ± 3

7 ± 3

26 ± 9

-

3

10 ± 5

141 ± 3

43 ± 4

7 ± 1

22 ± 3

-

10

11 ± 5

132 ± 21

47 ± 4

7 ± 2

26 ± 2

-

33

9 ± 1

152 ± 10

39 ± 7

9 ± 2

28 ± 3

-

100

10 ± 3

54 ± 10

38 ± 6

7 ± 2

22 ± 2

-

333

10 ± 3

58 ± 10

24 ± 2

9 ± 2

16 ± 1

-

1000

9 ± 1

56 ± 8

41 ± 1

8 ± 1

7 ± 3M,R

-

2500

13 ± 4

55 ± 3

28 ± 10

10 ± 2

6 ± 2M,R

-

5000

7 ± 3

38 ± 11M,R

37 ± 10

6 ± 0R

5 ± 2M,R

Positive controls, –S9

Name

NaN3

NaN3

MMS

4-NOPD

4-NOPD

Concentration

[μg/plate]

10

10

2.0 µL

50

10

Mean No. of colonies/plate

(average of 3 ± SD)

905 ± 35

2051 ± 72

671 ± 107

79 ± 8

374 ± 50

+

0 (DMSO)

12 ± 2

115 ± 8

47 ± 9

9 ± 3

36 ± 2

+

0

9 ± 4

190 ± 29

59 ± 11

11 ± 3

32 ± 7

+

3

12 ± 2

126 ± 34

51 ± 5

12 ± 2

34 ± 11

+

10

8 ± 2

119 ± 21

53 ± 6

13 ± 1

37 ± 11

+

33

13 ± 1

128 ± 11

52 ± 10

11 ± 2

36 ± 7

+

100

13 ± 2

110 ± 18

54 ± 8

9 ± 5

43 ± 9

+

333

13 ± 3

64 ± 18

56 ± 17

13 ± 2

40 ± 11

+

1000

7 ± 3

42 ± 6

57 ± 16

13 ± 1

33 ± 8

+

2500

8 ± 2

22 ± 9M,R

43 ± 4

8 ± 2M,R

13 ± 2M,R

+

5000

13 ± 1

2 ± 2M,R

45 ± 4

4 ± 2M,R

5 ± 1M,R

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentration

[μg/plate]

2.5

2.5

10

2.5

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

367 ± 24

3181 ± 117

421 ± 12

185 ± 12

3909 ± 258

NaN3: sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

M: manual count

R: reduced background growth

Conclusions:
Under the conditions of the conducted test the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation.
Executive summary:

The mutagenicity of the test item was studied with five mutant strains of Salmonella typhimurium

(TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) with and without metabolic activation according to OECD 471 under GLP conditions. Under the conditions of the conducted test the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
18 Sep - 06 Oct 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The substance was not tested in E. coli WP2 strains or S. thyphimurium TA102.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
yes
Remarks:
test strain with an AT base pair at the primary reversion site missing
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
other: TA1535, TA1537, TA1538, TA98 and TA100
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254 (500 mg/kg bw i.p.)
Test concentrations with justification for top dose:
Experiment 1 and 2
50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethylsulphoxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
dimethylsulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene (2-AA)
Remarks:
+S9: 2-AA (0.5 µg/plate TA535, TA1538, TA98 and TA100; 1 µg/plate TA1537); -S9: NaN3 (1 µg/plate, TA1535 and TA1535); 2-NF (1 µg/plate, TA98, TA1538); 9-AA (20 µg/plate, TA1537)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: microcolony growth
Evaluation criteria:
A test substance was considered as a mutagen if there was:
i) for S. typhimurium strains TA1535, TA1537, TA1538 and TA98, at least a doubling of the mean concurrent vehicle control values at some concentration of the test substances and, for S. typhimurium strain TA100, a 1.5-fold increase over the control value. If the mean colony count on the vehicle control plates was less than 10 then a value of 10 was assumed for assessment purposes. In such cases a minimum count of 20 was required before a significant mutagenic response was identified.
ii) a dose-related response, although at high dose levels this relationship could be inverted because of, for example, (1) toxicity to the bacteria, (2) specific toxicity to mutants and (3) inhibition of foreign compound metabolising enzymes where mutagens require metabolic activation by the liver.
iii) a reproducible effect in independent tests.
Statistics:
Mean values and standard deviations were calculated.
Species / strain:
other: TA1535, TA1537, TA1538, TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
starting at 1500 µg/plate in TA 98, TA 100, TA 1537 (exp 1 and 2) and TA 1538 (exp. 1) -S9; starting at 1500 µg/plate in TA 100 (exp 2) and TA 1538 (exp. 1) +S9; at 5000 µg/plate in TA 98 (exp. 2), TA 100 (exp. 1) and TA 1537 (exp. 1 and 2) +S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test substance was noted.

RANGE-FINDING/SCREENING STUDIES: A toxicity test using strain TA100 was performed in the presence and absence of S9 mix to establish suitable dose levels for the mutation tests. One plate for each of the following concentrations was used: 50, 150, 500, 1500 and 5000 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: The vehicle control values were within the normal ranges experienced in this laboratory and reported in the literature with these strains of S. typhimurium. The results obtained with the positive control substances were within the normal ranges expected for each bacterial strain and metabolic activation condition.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxicity to bacteria was noted at doses of 1500 and 5000 µg/plate, in the absence of S9 mix, in strains TA1537, TA1538, TA98 and TA100 (exp. 2). In the presence of S9 mix, toxicity to the bacteria was observed in strains TA1537 and TA100 at 5000 µg/plate and in strain TA1538 at dose levels of 1500 and 5000 µg/plate.

Table 1. Test results of experiment 1 (plate incorporation).

With or without S9-Mix

Test substance concentration

[μg/plate]

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA1535

TA100

TA1538

TA98

TA1537

0 (DMSO)

8 ± 4

89 ± 14

13 ± 4

20 ± 2

10 ± 1

50

11 ± 5

81 ± 15

9 ± 3

18 ± 2

9 ±2

150

12 ± 3

81 ± 17

12 ± 4

15 ± 7

9 ± 3

500

10 ± 2

86 ± 13

9 ± 3

19 ± 5

7 ± 1

1500

6 ± 2

82 ± 7#

8 ± 2#

15 ± 4#

12 ± 5#

5000

7 ± 2

74 ± 18#

9 ± 3#

19 ±4#

11 ± 1#

Positive controls, –S9

Name

NaN3

NaN3

2-NF

2-NF

9-AA

Concentration

[μg/plate]

1

1

1

1

20

Mean No. of colonies/plate

(average of 3 ± SD)

76 ± 19

422 ± 33

186 ± 28

142 ± 3

54 ± 14

+

0 (DMSO)

12 ± 2

95 ± 8

19 ± 2

29 ± 2

8 ± 2

+

50

10 ± 2

86 ± 5

20 ± 7

23 ± 6

11 ± 2

+

150

10 ± 5

81 ± 11

14 ± 4

22 ± 3

8 ± 3

+

500

10 ± 3

100 ± 9

12 ± 2

27 ± 3

13 ± 6

+

1500

9 ± 3

98 ± 4

17 ± 3#

23 ± 5

9 ± 3

+

5000

10 ± 2

96 ± 12#

17 ± 4#

24 ± 5

10 ± 2#

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentration

[μg/plate]

0.5

0.5

0.5

0.5

1

Mean No. of colonies/plate

(average of 3 ± SD)

46 ± 11

183± 26

75 ± 8

114 ± 8

36 ± 9

DMSO: dimethylsulphoxide

NaN3: sodium azide

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

2-NF: 2-nitrofluorene

#: thin lawn of microcolonies (i.e. toxicity)

 

Table 2. Test results of experiment 2 (plate incorporation).

With or without S9-Mix

Test substance concentration

[μg/plate]

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA1535

TA100

TA1538

TA98

TA1537

0 (DMSO)

8 ± 3

110 ± 10

19 ± 1

28 ± 10

9 ± 2

50

10 ± 3

108 ± 11

16 ± 2

21 ± 1

8 ± 1

150

10 ± 2

92 ± 15

15 ± 3

27 ± 2

7 ± 1

500

7 ± 2

75 ± 10

16 ± 4

26 ± 3

8 ± 1

1500

9 ± 5

94 ± 12#

15 ± 5

23 ± 4#

8 ± 2#

5000

8 ± 3

84 ± 7#

16 ± 1

25 ± 4#

7 ± 0#

Positive controls, –S9

Name

NaN3

NaN3

2-NF

2-NF

9-AA

Concentration

[μg/plate]

1

1

1

1

20

Mean No. of colonies/plate

(average of 3 ± SD)

174 ± 20

627 ± 52

209 ± 11

186 ± 14

177 ± 13

+

0 (DMSO)

7 ± 2

101 ± 11

22 ± 3

35 ± 4

8 ± 1

+

50

10 ± 2

101 ± 12

21 ± 2

30 ± 1

8 ± 1

+

150

9 ± 4

118 ± 4

19 ± 6

30 ± 8

7 ± 2

+

500

11 ± 3

104 ± 7

23 ± 3

25 ± 5

8 ± 0

+

1500

13 ± 1

100 ± 10

24 ± 1#

29 ± 2

9 ± 1

+

5000

8 ± 3

107 ± 11#

23 ± 3#

36 ± 4

7 ± 2#

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentration

[μg/plate]

0.5

0.5

0.5

0.5

1

Mean No. of colonies/plate

(average of 3 ± SD)

76 ± 19

189 ± 9

105 ± 2

142 ± 40

39 ± 3

DMSO: dimethylsulphoxide

NaN3: sodium azide

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

2-NF: 2-nitrofluorene

#: thin lawn of microcolonies (i.e. toxicity)

Conclusions:
Under the conditions of the conducted test the substance was not mutagenic in any of the five strains (TA1535, TA1537, TA1538, TA98 and TA100) tested with and without metabolic activation.
Executive summary:

The mutagenicity of the test item was studied with five mutant strains of Salmonella typhimurium

(TA 1535, TA 1537, TA 1538, TA 98 and TA 100) with and without metabolic activation according to OECD 471 under GLP conditions. Under the conditions of the conducted test the substance was not mutagenic in any of the five strains (

TA 1535, TA 1537, TA 1538, TA 98 and TA 10) tested with and without metabolic activation.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A bacterial gene mutation assay with the test substance was performed in accordance to OECD Guideline 471 and in compliance with GLP (2015). In this study the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvr A) tested with and without metabolic activation up to 5000 µg/plate.

An additional bacterial gene mutation assay with the test substance was performed similar to OECD Guideline 471 and in compliance with GLP (1985). In this study the substance was not mutagenic in any of the five strains (TA 1535, 1537, TA 1538, TA 98 and TA 100) tested with and without metabolic activation up to 5000 µg/plate.

Justification for classification or non-classification

The available experimental data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on the available data on genetic toxicity, the test item is not classified according to Regulation (EC) 1272/2008 (CLP), as amended for the eighth time in regulation (EU) No 2016/918.