Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test: Under the conditions of the study, it is concluded that the test material is not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay in both the presence and absence of metabolic activation.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 July 2016 to 01 August 2016
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
Type of assay:
bacterial forward mutation assay
Target gene:
- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid) and incubated in a shaking incubator (37 ± 1 °C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/mL). Freshly grown cultures of each strain were used for testing.
- Properly maintained: Yes. The Salmonella typhimurium strains are regularly checked to confirm their histidine requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV sensitivity and the number of spontaneous revertants. Stock cultures of the strains were stored in liquid nitrogen (-196 °C).
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Type and identity of media: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid) and incubated in a shaking incubator (37 ± 1 °C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/mL). Freshly grown cultures of each strain were used for testing.
- Properly maintained: Yes. The strain is regularly checked to confirm the tryptophan requirement, UV-sensitivity and the number of spontaneous revertants. Stock cultures were stored in liquid nitrogen (-196 °C).
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (rat liver S9-mix induced by Aroclor 1254)
Test concentrations with justification for top dose:
- Dose range finding study (TA100 and WP2uvrA only): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (absence and presence of S9-mix)
- Experiment 1 (TA1535, TA1537 and TA98): 0.55, 1.7, 5.4, 17, 52 and 164 μg/plate (absence and presence of S9-mix)
- Experiment 2 (all strains): 5, 10, 25, 50, 100 and 164 μg/plate (absence and presence of S9-mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Chosen following a solubility test
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DOSE RANGE FINDING TEST/ MUTATION ASSAY
Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without 5 % (v/v) S9-mix and reported as part of the first mutation experiment. The highest concentration of test material used in the subsequent mutation assay was the level at which the test material inhibited bacterial growth.

MUTATION ASSAY
At least five different doses (increasing with approximately half-log steps) of the test material were tested in each strain both in the absence and presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment with additional parameters, the test material was tested both in the absence and presence of 10 % (v/v) S9-mix in all tester strains.
Top agar in top agar tubes was melted by heating to 45 ± 2 °C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test material in DMSO and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

NUMBER OF REPLICATIONS: Testing was performed in triplicate

COLONY COUNTING
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test material precipitate to interfere with automated colony counting were counted manually. Evidence of test material precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

DETERMINATION OF CYTOTOXICITY
- Method: To determine the toxicity of the test material, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY
The assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at the testing facility.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5 % of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

DATA EVALUATION
In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test material is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
A test material is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Statistics:
No formal hypothesis testing was done..
Key result
Species / strain:
S. typhimurium, other: TA1535, TA1537, TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
DOSE RANGE FINDING TEST/FIRST MUTATION EXPERIMENT
- Precipitate: Precipitation of the test material on the plates was not observed at the start or at the end of the incubation period in any tester strain. Due to the colour of the test material the number of revertants and bacterial background lawn could not be determined at dose levels of 512 to 5000 μg/plate.
- Toxicity: Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn, was observed I the range finding test in TA100 (absence and presence of S9-mix) and WP2uvrA (absence of S9-mix). Cytotoxicity was observed in the first mutation experiment in all three tester strains in the absence and presence of S9-mix.
- Mutagenicity: No increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

MUTATION EXPERIMENT 2
- Precipitate: Precipitation of the test material on the plates was not observed at the start or at the end of the incubation period.
- Toxicity: Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn, was observed in all tester strains in the absence and presence of S9-mix.
- Mutagenicity: In the second mutation assay, no increase in the number of revertants was observed upon treatment with the test material under all conditions tested.
In strain TA100, a fluctuation in the mean number of revertant colonies above the laboratory historical control data range was observed in the absence of S9-mix. However, since the increase was not two-fold (a maximum of 1.2-fold was reached), this increase was not considered to be relevant.

DISCUSSION
All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two experiments.
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Table 1: Dose Range-finder and Experiment 1 (Plate incorporation assay 5 % S9)

+/- S9 Mix

Concentration (µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

PC

DMSO

0.55

1.7

5.4

17

52

164

512

1600

5000

1135

117

-

98

123

89 n

15 s

e NP MC

ND

ND

ND

866

10

11

15

22

25

11 n

5 m NP

-

-

-

1242

21

-

19

18

23

17

4 n NP

ND

ND

ND

1377

22

22

23

31

27

19 n

1 s NP

-

-

-

755

8

5

7

18

5 n

1 m

1 a NP

-

-

-

+

PC

DMSO

0.55

1.7

5.4

17

52

164

512

1600

5000

1230

99

-

96

140

126 n

94 s

e NP MC

ND

ND

ND

263

10

10

13

21

17 n

13 s

6 m NP

-

-

-

323

22

-

24

16

28

24

12 n NP

ND

ND

ND

1028

29

29

28

51

33

34 n

1 s NP

-

-

-

400

10

13

9

12

14 n

4 m

1 a NP

-

-

-

Mean number of revertant colonies/3 replicate plates

PC = Positive control

MC = Microcolonies

NP = No precipitate

ND = Not determined - the number of revertants and the bacterial background lawn could not be determined due to the colour of the test material

a = Bacterial background lawn absent

e = Bacterial background lawn extremely reduced

m = Bacterial background lawn moderately reduced

n = Normal bacterial background lawn

s = Bacterial background lawn slightly reduced

 

Table 2: Experiment 2 (Plate incorporation assay 10 % S9)

+/- S9 Mix

Concentration (µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

PC

DMSO

5

10

25

50

100

164

922

131

131

120

154 n

26 s

5 m

a NP MC

843

10

11

12

16

7 n

7 s

a NP MC

1516

19

24

18

11

9

1 n

0 a NP

1252

23

16

20

19

15

6 n

2 s NP

870

7

10

5

3 n

4 m

0 a

0 a NP

+

PC

DMSO

5

10

25

50

100

164

1155

109

114

119

155

127 n

48 s

e NP MC

136

13

17

13

11

10 n

4 s

e NP MC

483

36

32

28

20

24

12 n

0 e NP

556

32

22

37

29

23

23

4 n NP

499

12

9

11

18 n

8 s

4 m

a NP MC

Mean number of revertant colonies/3 replicate plates

PC = Positive control

MC = Microcolonies

NP = No precipitate

a = Bacterial background lawn absent

e = Bacterial background lawn extremely reduced

m = Bacterial background lawn moderately reduced

n = Normal bacterial background lawn

s = Bacterial background lawn slightly reduced

Conclusions:
Under the conditions of this study, it is concluded that the test material is not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions.

The test material was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).

In the dose range finding test, the test material was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test material did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn was observed in TA100 (absence and presence of S9-mix) and WP2uvrA (absence of S9-mix). Due to the colour of the test material the number of revertants and bacterial background lawn could not be determined at dose levels of 512 to 5000 μg/plate. Results were reported as part of the first mutation assay.

Based on the results of the dose range finding test, the test material was tested in the first mutation assay at a concentration range of 0.55 to 164 μg/plate in the absence and presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. Cytotoxicity was observed in all three tester strains in the absence and presence of S9-mix.

In a follow-up experiment of the assay with additional parameters, the test material was tested at a concentration range of 5 to 164 μg/plate in the absence and presence of 10 % (v/v) S9-mix in all strains. Cytotoxicity was observed in all tester strains in the absence and presence of S9-mix.

The test material did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains TA1535, TA1537, TA98 and TA100 and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Under the conditions of this study, it is concluded that the test material is not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

The test material was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).

In the dose range finding test, the test material was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test material did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn was observed in TA100 (absence and presence of S9-mix) and WP2uvrA (absence of S9-mix). Due to the colour of the test material the number of revertants and bacterial background lawn could not be determined at dose levels of 512 to 5000 μg/plate. Results were reported as part of the first mutation assay.

Based on the results of the dose range finding test, the test material was tested in the first mutation assay at a concentration range of 0.55 to 164 μg/plate in the absence and presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. Cytotoxicity was observed in all three tester strains in the absence and presence of S9-mix.

In a follow-up experiment of the assay with additional parameters, the test material was tested at a concentration range of 5 to 164 μg/plate in the absence and presence of 10 % (v/v) S9-mix in all strains. Cytotoxicity was observed in all tester strains in the absence and presence of S9-mix.

The test material did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains TA1535, TA1537, TA98 and TA100 and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Under the conditions of this study, it is concluded that the test material is not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.