Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria: Key study: According to OECD 471 Guideline. GLP study. The test item was found to be mutagenic for the strain TA100 under the test conditions of pre-incubation.

In vitro gene mutation study in bacteria: Key study: According to OECD 471 Guideline. GLP study. Under the experimental conditions reported, the test item was found to be mutagenic for the strain TA100.

In vitro gene mutation study in bacteria: Key study: According to OECD 471 Guideline. GLP study. Under the experimental conditions reported, the test item was found to be mutagenic for strains TA100 and TA 102.

In vitro gene mutation study in bacteria: Key study: According to OECD 471 Guideline. GLP study. Under the experimental conditions reported, the test item was found to be mutagenic for the strain TA100.

In vitro cytogenicity / chromosome aberration study in mammalian cells: Experimental study on-going. According to OECD 473 Guideline. GLP study. Results are not available yet.

In vitro gene mutation study in mammalian cells: Experimental study on-giong. According to OECD 490 Guideline. GLP study. Results are not available yet.

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 08 August 2016 to 22 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)
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
Target gene:
Histidine-requiring gene in Salmonella typhimurium
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: uvrB and rfa mutated
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: rfa mutated
Metabolic activation:
with and without
Metabolic activation system:
S9 mix of liver rats
Test concentrations with justification for top dose:
5000, 1600, 500, 160 and 50 μg/plate. The preliminary study showed no cytotoxicity of the test item at the highest dose tested (5000 μg/plate); therefore this concentrations range was used for the mutagenicity test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionized water. - Justification for choice of solvent/vehicle: A preliminary dissolution test was performed in order to define the most appropriate solvent as well as the maximal concentration tested.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION:: - Direct plate incorporation method (Test 1): a first experiment of genotoxicity was conducted, with and without metabolic activation, on the range of concentrations defined by the preliminary study: 2 ml of top agar, 0.1 ml of bacterial inoculum and 0.1 ml of the test item with/without S9-mix (0.5 ml of S9-mix or 0.5 ml buffer) were mixed and poured on the surface of the bottom agar. After solidification, plates were incubated at 37 °C ± 2 °C during 48 to 72 hours. Positive and negative controls were included in the experiment. After the mentioned period, colonies were counted. - Pre-incubation method (Test 2): a second experiment was performed, also with and without metabolic activation, with dose levels defined after analysis of results obtained on the first experiment. This second experiment was performed in order to confirm or for complement results of the first one: 0.1 ml of bacterial inoculum and 0.1 ml of test item with/without S9-mix (0.5 ml of S9-mix or 0.5 ml buffer) were mixed and incubated at 37ºC ± 2 °C to 30 min. After this period, 2 ml of top agar was added and mixed. The mixture was poured on the surface of the bottom agar and plates were incubated at 37 °C ± 2 °C during 48 to 72 hours. Positive and negative controls were included in the experiment. After the mentioned period, colonies were counted.- Cell density at seeding: it was applied 0.1ml of bacterial inoculum containing 10^8-9 cells/ml.DURATION- Preincubation period: about 20-30min at 37ºC (+/-2ºC), only for the Test 2- Exposure duration: 48-72hSELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.NUMBER OF REPLICATIONS: 3DETERMINATION OF CYTOTOXICITYPetri plates were observed and any cytotoxicity sign was reported (bottom bacterial layer reduction). The cytotoxicity intensity on the bottom bacterial layer is evaluated qualitatively on each plate by naked eyes:a. total destruction of the bottom bacterial layer (the revertants development does not occurs in this case).b. moderated destruction of the bottom bacterial layer.In the preliminary experiment is considered the test item as cytotoxic if the spontaneous revertant rate is lower than 0.7 (R < 0.7). The possible destruction of the bottom bacterial layer is also taken into account.OTHER:- Methodology of the preliminary experiment (direct plate incorporation method): it was performed on the strain S. typhimurium TA100 in order to evaluate the cytotoxicity of the test item and to select the range of dose levels for the further experiments: Top agar, buffer, bacterial inoculum and the test item with/without S9-mix were mixed and poured on the surface of the bottom agar. After solidification, plates were incubated at 37 °C ± 2 °C during 48 to 72 hours. Positive and negative controls were included in the experiment. After the mentioned period, colonies were counted.- Method of counting: All colony numbers were determined by a plate reader (Sorcerer, v 2.2)
Evaluation criteria:
The test item is considered as mutagenic if at the end of the verifications steps, it has been obtained, in a reproducible way, a relation dose-effect on one or some of 5 strains with and/or without metabolic activation. The mutagenicity is taken into account for a given concentration only when the number of revertants is equal at least to the double of the spontaneous rate of reversion for TA98, TA100 and TA102 strains (R >= 2) and the triple of the spontaneous rate of reversion for TA1535 and TA1537 strains (R >= 3).The test item is considered as non-mutagenic if, in the outcome at the Test 1 and the Test 2, the rate of revertants always remained lower than the double of the rate of spontaneous reversion for all the concentrations of tested product, for TA98, TA100 and TA102 strains (R < 2) and the triple of the spontaneous rate of reversion for TA1535 and TA1537 strains (R < 3), with and without metabolic activation, and on the condition of having made sure that the absence of mutagen effect is not bound to the toxicity of the experimented concentrations.
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
at 5000 μg/plate
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS- Precipitation: no sign of precipitate was observed.RANGE-FINDING/SCREENING STUDIES: The preliminary cytotoxicity test performed on the TA100 strain showed no cytotoxicity of the test item at the highest dose tested (5000 μg/plate) (R >= 0.7). The same range of concentrations was used for the Test 1 and 2.HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)- Positive historical control data:Without S9TA98: range 746.3-1531.3, mean 1095.7, SD 163.5 (n=77)TA100: range 1663.0-3329.7, mean 2131.4, SD 288.4 (n= 80)TA102: range 1737.7-3921.3, mean2737.5, SD 578.3 (n=71)TA1535: range 1659.3-2803.3, mean2154.9, SD 226.7 (n=73)TA1537: range 117.7-1082.0 , mean 333.1, SD 196.0 (n=74) With S9TA98: range 931.0-3686.3 , mean 2047.5, SD755.5 (n=77)TA100: range 923.3-4667.0, mean 2592.7, SD 988.7 (n=76) TA102: range 1955.7-6017.3, mean 3631.8, SD 799.6 (n=72)TA1535: range 151.0-433.7, mean 250.1, SD 76.5 (n=73)TA1537:range 134.7-506.7, mean 275.7, SD 101.6 (n=75)- Negative (solvent/vehicle) historical control data:Without S9TA98: range 16.7-42.0, mean 26.1, SD 4.2 (n=192)TA100: range 49.7-220.3, mean 142.8, SD 43.9 (n=) TA102: range 275.3-461.7, mean 370.9, SD 29.3 (n=183)TA1535: range 10.0-38.0, mean 21.2, SD 5.9 (n=183)TA1537:range 9.3-28.0, mean 17.9, SD 3.8 (n=189)With S9TA98: range 24.7-56.7, mean 34.7, SD 5.3 (n=194)TA100: range 58.3-233.0, mean 164.8, SD 44.4 (n=198) TA102: range 331.7-526.7, mean 433.7, SD 36.2 (n=179)TA1535: range 12.7-38.3, mean 21.5, SD 5.9 (n=182)TA1537:range 12.0-37.0, mean 22.9, SD 4.4 (n=186)The positive controls must show a revertants number equal at least to the double of the spontaneous rate of reversion for TA98, TA100 and TA102 (R >= 2) and the triple of the spontaneous rate of reversion for TA1535 and TA1537 (R >=3).The mean negative controls are within the historical data.All criteria of validity regarding control plates were met.ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxic effect was observed. Ratios 0.6 shown during Test 1 on strain TA1535 at 160 and 50 μg/plate without metabolic activation must not be considered according ratios observed at the upper concentrations.
Remarks on result:
other: Test 2

TEST 1: Results for each strain and dose

  

Concentrations (μg/plate)

Mean values of revertants / Ratio treated – solvent (R)

Salmonella Thypimurium strains

 

 

TA 100

TA 102

TA1535

TA 1537

TA 98

 

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

5000

Mean

283.3

259.3

436.7

432.0

19.3

19.0

19.0

28.3

47.3

49.0

R

1.5

1.3

1.1

1.0

0.8

1.1

1.1

0.9

1.3

1.3

1600

Mean

207.3

235.0

397.3

381.0

17.0

16.7

25.3

23.0

40.3

40.7

R

1.1

1.1

1.0

0.9

0.7

1.0

1.5

0.8

1.1

1.1

500

Mean

193.3

202.3

378.3

425.0

18.0

18.7

15.3

25.0

35.3

46.7

R

1.0

1.0

0.9

1.0

0.8

1.1

0.9

0.8

1.0

1.3

160

Mean

191.3

195.7

359.7

375.3

13.0

17.7

16.3

20.3

38.0

38.3

R

1.0

0.9

0.9

0.9

0.6

1.0

1.0

0.7

1.0

1.0

50

Mean

187.3

235.0

368.7

411.3

13.7

16.0

22.0

20.7

33.3

45.7

R

1.0

1.1

0.9

0.9

0.6

0.9

1.3

0.7

0.9

1.2

PC

Mean

1909.7

4156.3

2501.7

3854.7

2549.7

292.7

798.7

494.7

1095.0

3646.7

 

R

10.1

20.0

6.2

8.8

110.9

16.9

47.0

16.3

29.6

98.6

NC (Water)

Mean

188.3

207.3

406.7

439.7

23.0

17.3

17.0

30.3

37.0

37.0

NC(untreated)

Mean

183.7

182.3

362.7

449.3

17.0

15.0

13.7

29.0

24.0

37.7

 

 

TEST 2: Results for each strain and dose

  

Concentrations (μg/plate)

Mean values of revertants / Ratio treated – solvent (R)

Salmonella Thypimurium strains

 

 

TA 100

TA 102

TA1535

TA 1537

TA 98

 

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

5000

Mean

416.7

299.0

444.7

593.3

24.7

30.3

14.3

21.0

36.0

40.0

R

2.5

1.4

1.2

1.4

1.5

2.0

0.9

1.0

1.4

1.4

1600

Mean

195.7

248.0

397.3

489.7

19.0

17.7

19.3

14.0

28.7

34.7

R

1.2

1.2

1.1

1.1

1.1

1.2

1.2

0.7

1.1

1.2

500

Mean

172.3

197.3

382.3

455.0

17.3

20.7

13.3

18.3

18.3

33.7

R

1.0

0.9

1.1

1.0

1.0

1.3

0.8

0.9

0.7

1.2

160

Mean

154.7

179.3

359.3

439.3

14.3

17.7

18.3

17.7

20.7

28.7

R

0.9

0.9

1.0

1.0

0.9

1.2

1.1

0.9

0.8

1.0

50

Mean

176.0

187.3

368.0

434.3

13.7

16.7

16.3

24.0

23.7

42.0

R

1.0

0.9

1.0

1.0

0.8

1.1

1.0

1.2

0.9

1.5

PC

Mean

1949.7

2424.0

3026.7

3737.7

2221.7

220.0

752.0

279.7

914.0

2116.7

 

R

11.5

11.6

8.3

8.6

133.3

14.3

46.0

13.5

34.7

74.7

NC (Water)

Mean

169.3

209.0

364.0

435.3

16.7

15.3

16.3

20.7

26.3

28.3

NC(untreated)

Mean

165.7

200.3

342.0

420.0

17.0

17.3

18.0

21.0

20.7

26.7

   

PC = positive control

NC = negative control

Conclusions:
The test item was found to be mutagenic for strain TA100 under the test conditions of pre-incubation.
Executive summary:

The aim of this test was to determine the ability of the test item to induce mutation, it was assessed using the bacterial reverse mutation test (Ames test). The test was performed following the OECD Guideline 471 with GLP on five Salmonella typhimurium strains. The test item dilutions were prepared in water for analysis. Firstly, a preliminary cytotoxicity test was performed on S. typhimurium TA100 strain with the following concentrations: 5000, 1600, 500, 160 and 50μg/plate, for triplicate, with and without S9. Plates were incubated at 37ºC for 48-72h and after this period colonies were counted. As the preliminary study showed no cytotoxicity of the test item, this concentrations range was used for the genotoxicity Test 1 (direct method) and Test 2 (pre-incubation method), and positive and negative/solvent controls were included. The same test conditions as the preliminary test were used. The revertant analysis showed no cytotoxic effect. Moreover, it was observed a ratio R higher to the double of the spontaneous rate of reversion during Test 2 for strain TA100 at 5000μg/plate without metabolic activation: R = 2.5. Besides, a dose response was observed during Test 2 for this strain TA100 without metabolic activation, and for strain TA1535 with metabolic activation. In addition, no sign of precipitate was observed. Based on the result of this study, the test item was found to be mutagenic for strain TA100 under the test conditions of pre-incubation.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 03 December 2015 to 18 January 2016
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
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine-requiring gene in Salmonella typhimurium
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: his D 3052; rfa-: uvrB-: R-factor
Remarks:
frame shift mutations
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: his G 46; rfa-: uvrB-: R-factor
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
other: his G 46; rfa-: uvrB-
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
other: his C 3076; rfa-: uvrB-
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: his G 428 (pAQ1); rfa-: R-factor
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction (rat)
Test concentrations with justification for top dose:
The test item was tested in the pre-experiment with the following concentrations: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate.The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment in which no cytotoxicity was observed at the highest dose tested. (5000 μg/plate). The concentration range covered two logaritmic decades. Two independent experiments were performed with the following concentrations: 31.6, 100, 316, 1000, 2500 and 5000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: A. dest., Eurofins Munich, Lot No. 151125 and 151230. - Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine and 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: - Direct plate incorporation method (Test 1): 100 μl of the test solution at each dose level, 100 μl of bacterial suspension and 2000 μl of overlay agar with/without S9 mix (500 μl of S9-mix or buffer) were mixed in a test tube and poured over the surface of a minimal agar plate. For each strain and dose level, including the controls, thee plates were used. After solidification, the plates were inverted and incubated at 37ºC for at least 48 h in the dark.- Pre-incubation method (Test 2): 100 μl of the test solution were pre-incubated with 100 μl of bacterial suspension with/without S9 mix (500 μl of S9-mix or buffer) for 60 min at 37ºC, prior to adding 2000 μl of overlay agar and pouring onto the surface of a minimal agar plate. For each strain and dose level, including the controls, thee plates (except for one case, in which two plates were evaluated) were used. After solidification, the plates were inverted and incubated at 37ºC for at least 48 h in the dark.- Cell density at seeding: 100 μl bacterial suspension where 10^9 cells/mLDURATION- Preincubation period: 60 min at 37ºC (test 2)- Exposure duration: at least 48h at 37ºC in the dark.SELECTION AGENT (mutation assays):the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.NUMBER OF REPLICATIONS: 3 (except for one case, in which two plates were evaluated: Experiment 2, tester strain TA 102, dose 31.6 μg/plate with S9).DETERMINATION OF CYTOTOXICITYThe cytotoxicity could be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately <= 0.5 in relation to the solvent control.OTHER:- Preliminary experiment: (Direct plate incorporation method): Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as described for the main experiment I.- Method of counting: the colonies were counted using a ProtoCOL counter. If precipitation of the test item precluded automatic counting the revertant colonies were counted by hand. In addition, tester strains with a low spontaneous mutation frequency like TA 1535 and TA 1537 were counted manually.
Evaluation criteria:
Evaluation of MutagenicityThe Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).A test item is considered as mutagenic if:- a clear and dose-related increase in the number of revertants occurs and/or- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.A biologically relevant increase is described as follows:- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
at 5000 μg/plate, experiment 1
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 μg/plate, experiment 2
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
at 5000 μg/plate, experiment 2
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 μg/plate, in experiment 1
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS- Precipitation: No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).RANGE-FINDING/SCREENING STUDIES: No cytotoxicity was detected, the mutation factor was >0.5. The highest dose used for the range finding study was also used for main experiments.HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)- Positive historical control data:Without S9TA 98: mean 443.7, SD 183.1, range 192-2213 (n= 1172)TA 100: mean 704.8, SD 272.7, range 132-1498 (n= 1285)TA 1535: mean 858.3, SD 320.2, range 34-1472 (n= 1042)TA 1537: mean 93.2, SD 27.3, range 30-273 (n= 1054)TA 102: mean 1733.5, SD 408.3, range 162-3181 (n= 688)With S9TA 98: mean 2318.0, SD 573.6, range 128-3606 (n= 1156)TA 100: mean 1839.6, SD 455.1, range 169-3132 (n= 1284)TA 1535: mean 100.0, SD 60.6, range 19-1011 (n= 1041)TA 1537: mean 218.6, SD 85.2, range 28-489 (n= 1039)TA 102: mean 663.9, SD 176.6, range 137-2001 (n= 688)- Negative (solvent/vehicle) historical control data:Without S9TA 98: mean 22.3, SD 4.8, range 13-48 (n= 1159)TA 100: mean 95.5, SD 18.1, range 61-182 (n= 1281)TA 1535: mean 10.9, SD 5.1, range 4-35 (n= 1043)TA 1537: mean 7.5, SD 2.5, range 2.-27 (n= 1043)TA 102: mean 230.5, SD 47.8, range 136-415 (n= 684)With S9TA 98: mean 29.9, SD 6.2, range 13-61 (n= 1157)TA 100: mean 103.4, SD 17.4, range 68-194 (n= 1286)TA 1535: mean 9.1, SD 3.3, range 4-34 (n= 1042)TA 1537: mean 7.6, SD 2.7, range 3-31 (n= 1040)TA 102: mean 290.9, SD 65.3, range 91-495 (n= 683)All criteria of validity regarding control plates were met.ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activation) in experiment I and II, except tester strain TA1537 in experiment I and tester strain TA 100 in experiment II at a concentration of 5000 μg/plate (both without metabolic activation).

Test 1: Results for each strain and dose

Concentrations (μg/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella Thypimurium strains

 

 

TA 100

TA 102

TA1535

TA 1537

TA 98

 

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

31.6

Mean

100

95

242

308

9

9

7

9

49

55

MF

1.2

1.0

1.1

1.2

0.9

0.9

0.8

1.2

0.9

1.3

100

Mean

86

96

205

310

5

5

8

6

44

44

MF

1.0

1.0

0.9

1.2

0.5

0.5

0.9

0.8

0.8

1.0

316

Mean

82

99

229

278

7

9

6

8

45

40

MF

1.0

1.0

1.0

1.1

0.7

0.9

0.7

1.0

0.8

0.9

1000

Mean

113

109

256

292

9

9

7

5

39

37

MF

1.4

1.1

1.1

1.2

0.9

0.8

0.8

0.7

0.7

0.9

2500

Mean

131

93

253

335

14

9

8

4

45

45

MF

1.6

1.0

1.1

1.3

1.4

0.9

1.0

0.6

0.8

1.0

5000

Mean

167

152

241

330

10

13

4

6

51

52

MF

2.0

1.6

1.1

1.3

1.0

1.3

0.5

0.8

0.9

1.2

PC – I

Mean

501

-

1116

-

501

-

90

-

413

-

MF

6.0

-

4.9

-

50.1

-

10.3

-

7.6

-

PC – II

Mean

-

1607

-

901

-

104

-

297

-

2788

MF

-

16.5

-

3.6

-

10.1

-

38.8

-

64.3

NC A. dest.

Mean

83

97

228

253

10

10

9

8

54

43

MF

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

Test 2: Results for each strain and dose

Concentrations (μg/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella Thypimurium strains

 

 

TA 100

TA 102

TA1535

TA 1537

TA 98

 

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

31.6

Mean

65

77

323

380

14

5

10

8

42

34

MF

0.8

0.9

1.2

1.0

1.7

0.6

1.3

1.8

1.0

1.0

100

Mean

72

95

336

351

12

9

10

7

43

30

MF

0.9

1.1

1.2

1.0

1.4

0.9

1.3

1.6

1.0

0.9

316

Mean

47

99

352

371

7

5

9

6

41

35

MF

0.6

1.2

1.3

1.0

0.8

0.5

1.1

1.4

1.0

1.1

1000

Mean

91

110

381

381

9

9

10

9

39

38

MF

1.1

1.3

1.4

1.0

1.1

1.0

1.3

2.1

0.9

1.2

2500

Mean

131

126

378

518

8

8

8

9

26

29

MF

1.6

1.5

1.4

1.4

1.0

0.9

1.0

2.2

0.6

0.9

5000

Mean

40

177

422

660

5

12

7

10

30

31

MF

0.5

2.1

1.6

1.8

0.6

1.2

0.8

2.2

0.7

0.9

PC – I

Mean

419

-

882

-

689

-

109

-

447

-

MF

5.2

-

3.3

-

82.7

-

13.6

-

10.7

-

PC – II

Mean

-

1451

-

823

-

90

-

103

-

1983

MF

-

17.3

-

2.3

-

9.3

-

23.8

-

60.7

NC A. dest.

Mean

81

84

270

364

8

10

8

4

42

33

MF

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

PC = positive control

NC = negative control

PC – I = 4-NOPD, MMS or NaN3

Conclusions:
Under the experimental conditions reported, the test item was found to be mutagenic for strain TA100.
Executive summary:

The aim of this test was to determine the ability of the test item to induce mutation, it was assessed using the bacterial reverse mutation test (Ames test). The test was performed following the OECD Guideline 471 with GLP on five Salmonella typhimurium strains. Firstly, a preliminary cytotoxicity test was performed on S. typhimurium TA 98 and TA100 strain with the following concentrations:3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000μg/plate, for triplicate, with and without S9. Plates were incubated at 37ºC for at least 48h in the dark and after this period colonies were counted. The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 5000μg/plate was selected as the maximum concentration. Two independent experiments were performed with the following concentrations: 31.6, 100, 316, 1000, 2500 and 5000μg/plate and positive and negative/solvent controls (distilled water) were included. No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation). No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated in experiment I and II, except tester strain TA1537 in experiment I and tester strain TA 100 in experiment II at a concentration of 5000 μg/plate (both without metabolic activation). Biologically relevant increases of revertant colony numbers were observed in tester strain TA 100 at a concentration of 5000 μg/plate in experiment I (without metabolic activation) and at a concentration of 5000 μg/plate in experiment II (with metabolic activation). The threshold value of 2.0 was exceeded and a maximum mutation factor of 2.1 was reached at a concentration of 5000 μg/plate (with metabolic activation). In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item caused gene mutations by base pair changes in the genome of the tester strain TA 100.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 06 April 2016 to 15 April 2016
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
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine-requiring gene in Salmonella typhimurium.
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: his D 3052; rfa-: uvrB-: R-factor
Remarks:
frame shift mutations
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: his G 46; rfa-: uvrB-: R-factor
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
other: his G 46; rfa-: uvrB-
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
other: his C 3076; rfa-: uvrB-
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: his G 428 (pAQ1); rfa-: R-factor
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction (rat)
Test concentrations with justification for top dose:
The performance of a pre-experiment for toxicity was not regarded as necessary. 5.0 μL/plate was selected as the maximum concentration. Two independent experiments were performed with the following concentrations: 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate. The concentration range covered two logarithmic decades.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: A. dest., Eurofins Munich, Lot No. 160212. - Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: - Direct plate incorporation method (Experiment 1): 100 μl of the test solution at each dose level, 100 μl of bacterial suspension and 2000 μl of overlay agar with/without S9 mix (500 μl of S9-mix or buffer) were mixed in a test tube and poured over the surface of a minimal agar plate. For each strain and dose level, including the controls, thee plates were used. After solidification, the plates were inverted and incubated at 37ºC for at least 48 h in the dark.- Pre-incubation method (Experiment 2): 100 μl of the test solution were pre-incubated with 100 μl of bacterial suspension with/without S9 mix (500 μl of S9-mix or buffer) for 60 min at 37ºC, prior to adding 2000 μl of overlay agar and pouring onto the surface of a minimal agar plate. For each strain and dose level, including the controls, thee plates were used. After solidification, the plates were inverted and incubated at 37ºC for at least 48 h in the dark.- Cell density at seeding (if applicable): 100 μl bacterial suspension where 10^9 cells/mLDURATION- Preincubation period: 60 min at 37ºC (Experiment 2)- Exposure duration: at least 48h at 37ºC in the dark (inverted plates).SELECTION AGENT (mutation assays):the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize an essential amino acid.NUMBER OF REPLICATIONS: 3DETERMINATION OF CYTOTOXICITY- Method: Cytotoxicity could be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately <= 0.5 in relation to the solvent control.OTHER- Method of counting: the colonies were counted using a ProtoCOL counter. If precipitation of the test item precluded automatic counting the revertant colonies were counted by hand. In addition, tester strains with a low spontaneous mutation frequency like TA 1535 and TA 1537 were counted manually.
Evaluation criteria:
Evaluation of cytotoxicityCytotoxicity can be detected by a clearing rather disminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximatelly <= 0.5 in relation to the solvent control.Evaluation of MutagenicityThe Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).A test item is considered as mutagenic if:- a clear and dose-related increase in the number of revertants occurs and/or- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.A biologically relevant increase is described as follows:- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
at 5.0 μl/plate experiment 1 and 1 μl/plate experiment 2
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
2,5 μg/plate and higher, experiment 2
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
at 1 μl/plate and higher experiment 2
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
5.0 μL/plate, experiment 2
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
2.5 μL/plate and higher, experiment 2
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5.0 μL/plate, experiment 2
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
2.5 μL/plate and higher, experiment 2
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5.0 μL/plate, experiment 2
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS- Precipitation: No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)- Positive historical control data:Without S9TA 98: mean 444.5, SD 180.0, range 141-2213 (n= 1048)TA 100:mean 627.6, SD 255.3, range 132-1498 (n= 1195)TA 1535: mean 795.5, SD 323.7, range 34-1854 (n= 943)TA 1537: mean 92.0, SD 24.8, range 32-273 (n= 954)TA 102: mean 1801.0, SD 483.5, range 162-3321 (n=625)With S9TA 98: mean 2126.7, SD 679.0, range 113-3606 (n= 1032)TA 100:mean 1786.6, SD 494.6, range 169-3132 (n= 1194)TA 1535: mean 103.4, SD 64.9, range 20-1384 (n= 942)TA 1537: mean 221.8, SD 92.2, range 26-489 (n= 940)TA 102: mean 748.8, SD 176.3, range 137-1520 (n= 625)- Negative (solvent/vehicle) historical control data:Without S9TA 98: mean 23.1, SD 6.1, range 13-54 (n= 1035)TA 100: mean 89.2, SD 13.2, range 49-139 (n= 1190)TA 1535: mean 12.0, SD 5.8, range 4-39 (n= 944)TA 1537: mean 7.4, SD 2.6, range 2-35 (n= 943)TA 102: mean 251.4, SD 55.6, range 141-472 (n= 621)With S9TA 98: mean 29.9, SD 7.0, range 13-61 (n= 1033)TA 100:mean 98.3, SD 14.2, range 67-162 (n= 1196)TA 1535: mean 9.4, SD 3.7, range 4-32 (n= 943)TA 1537: mean 7.4, SD 2.8, range 3-36 (n= 941)TA 102: mean 317.7, SD 76.6, range 91-586 (n= 619)All criteria of validity regarding control plates were met.ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activatiob) in Experiment 1.In Experiment 2 toxic effects were noted in tester strains TA 98 and TA 1537 at a concentration of 5.0 μl/plate (without metabolic activation). In tester strains TA 100 and TA 1535 toxic effects of the test item were noted at concentrations of 2.5 μl/plate and higher (without metabolic activation).

Experiment 1: Results for each strain and each dose

Concentrations (μl/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella Thypimurium strains

 

 

TA 100

TA 102

TA1535

TA 1537

TA 98

 

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

0.0316

Mean

131

119

395

411

22

14

6

6

17

19

MF

1.1

1.2

 

0.8

1.0

1.0

1.1

1.4

0.9

0.7

0.100

Mean

135

118

416

491

20

15

6

6

21

28

MF

1.2

1.2

 

1.0

1.0

1.1

1.1

1.4

1.1

1.1

0.316

Mean

113

125

397

496

25

13

9

5

26

31

MF

1.0

1.3

 

1.0

1.2

0.9

1.6

1.2

1.4

1.2

1.0

Mean

117

125

361

473

29

21

4

7

19

26

MF

1.0

1.3

 

0.9

1.4

1.5

0.8

1.6

1.0

1.0

2.5

Mean

177

163

350

497

21

16

9

11

22

28

MF

1.5

1.6

 

1.0

1.0

1.2

1.6

2.5

1.2

1.1

5.0

Mean

237

191

402

575

20

21

8

10

27

28

MF

2.1

1.9

 

1.1

0.9

1.5

1.5

2.2

1.4

1.1

PC – I

Mean

1021

-

1823

-

1180

-

81

-

309

-

MF

8.9

-

 

-

55.3

-

14.2

-

16.2

-

PC – II (2-AA)

Mean

-

2622

-

1046

-

231

-

403

-

2173

MF

-

36.3

 

2.1

-

16.5

-

93.0

-

84.7

NC A. dest.

Mean

115

100

368

509

21

14

6

4

19

26

MF

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

Experiment 2: Results for each strain and each dose

Concentrations ( μl/plate )

Mean values of revertants / Mutation factor (MF)

Salmonella Thypimurium strains

 

 

TA 100

TA 102

TA1535

TA 1537

TA 98

 

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

0.0316

Mean

80

90

271

343

19

11

10

9

28

31

MF

0.9

1.2

1.0

1.0

0.9

1.1

1.0

1.0

1.1

1.0

0.100

Mean

81

98

222

351

24

11

7

8

28

35

MF

0.9

1.3

0.8

1.0

1.1

1.1

0.7

0.8

1.1

1.1

0.316

Mean

129

105

259

325

22

11

8

11

30

34

MF

1.4

1.4

0.9

1.0

1.0

1.1

0.8

1.1

1.2

1.1

1.0

Mean

373

153

327

436

22

13

8

11

35

39

MF

4.2

2.1

1.2

1.3

1.0

1.3

0.7

1.2

1.3

1.2

2.5

Mean

97

279

502

667

13

23

10

12

23

38

MF

1.1

3.8

1.8

2.0

0.6

2.3

0.9

1.3

0.9

1.2

5.0

Mean

50

557

707

992

10

17

6

9

15

57

MF

0.6

7.5

2.6

3.0

0.4

1.7

0.5

1.0

0.6

1.8

PC – I

Mean

486

-

1723

-

1192

-

113

-

411

-

MF

5.4

-

6.3

-

55.0

-

10.6

-

15.6

-

PC – II

Mean

-

1402

-

790

-

198

-

312

-

2117

MF

-

18.9

-

2.4

-

19.8

-

33.4

-

66.2

NC A. dest.

Mean

90

74

274

336

22

10

11

9

26

32

MF

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

PC = positive control

NC = negative control

PC – I = 4-NOPD, MMS or NaN3

Conclusions:
Under the experimental conditions reported, the test item was found to be mutagenic for strains TA100 and TA 102
Executive summary:

The aim of this test was to determine the ability of the test item to induce mutation, it was assessed using the bacterial reverse mutation test (Ames test). The test was performed following the OECD Guideline 471 with GLP on five Salmonella typhimurium strains. The performance of a pre-experiment for toxicity was not regarded as necessary. Two experiments were performed: experiment 1 with the plate incorporation method and the experiment 2 with the pre-incubation method. The following concentrations of the test item were used in these experiments: 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate. Additionally, positive and negative/solvent controls were included. After that, plates were incubated at 37ºC for at least 48h in the dark and after this period colonies were counted. No precipitation of the test item was observed in any tester strain used in experiment I and II. In experiment II toxic effects of the test item were noted in tester strains TA 98 and TA 102 at a concentration of 5.0 μL/plate (without metabolic activation). In tester strains TA 100 and TA 1535 toxic effects of the test item were noted at concentrations of 2.5 μL/plate and higher (without metabolic activation). On the other hand, Biologically relevant increases of revertant colony numbers were observed in tester strain TA 100 at a concentration of 5.0 μL/plate in experiment I (without metabolic activation) and in experiment II at a concentration of 1.0 μL/plate (without metabolic activation) and at concentrations of 1.0 μL/plate and higher (with metabolic activation). As well as biologically relevant increase of revertant colony numbers was observed in experiment II in tester strain TA 102 at a concentration of 5.0 μL/plate (without metabolic activation) and at concentrations of 2.5 μL/plate and higher (with metabolic activation).The threshold value of 2.0 was exceeded and a maximum mutation factor of 7.5 was reached in tester strain TA 100 at a concentration of 5.0 μL/plate (with metabolic activation, experiment II). In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item caused gene mutations by base pair changes in the genome of the tester strains TA 100 and TA 102.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 31 May 2016 to 10 June 2016
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
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine-requiring gene in Salmonella typhimurium
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: his D 3052; rfa-: uvrB-: R-factor
Remarks:
frame shift mutations
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: his G 46; rfa-: uvrB-: R-factor
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
other: his G 46; rfa-: uvrB-
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
other: his C 3076; rfa-: uvrB-
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: his G 428 (pAQ1); rfa-: R-factor
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction (rat)
Test concentrations with justification for top dose:
The performance of a pre-experiment for toxicity was not regarded as necessary. 5000 μg/plate was selected as the maximum concentration. Two independent experiments were performed with the following concentrations: 31.6, 100, 316, 1000, 2500 and 5000 μg/plate The concentration range covered two logarithmic decades.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: A. dest., Eurofins Munich, Lot No. 160510. - Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: - Direct plate incorporation method (Experiment 1): 100 μl of the test solution at each dose level, 100 μl of bacterial suspension and 2000 μl of overlay agar with/without S9 mix (500 μl of S9-mix or buffer) were mixed in a test tube and poured over the surface of a minimal agar plate. For each strain and dose level, including the controls, thee plates (except for one case, in which two plates were evaluated) were used. After solidification, the plates were inverted and incubated at 37ºC for at least 48 h in the dark.- Pre-incubation method (Experiment 2): 100 μl of the test solution were pre-incubated with 100 μl of bacterial suspension with/without S9 mix (500 μl of S9-mix or buffer) for 60 min at 37ºC, prior to adding 2000 μl of overlay agar and pouring onto the surface of a minimal agar plate. For each strain and dose level, including the controls, thee plates were used. After solidification, the plates were inverted and incubated at 37ºC for at least 48 h in the dark.- Cell density at seeding (if applicable): 100 μl bacterial suspension where 10^9 cells/mLDURATION- Preincubation period: 60 min at 37ºC (test 2)- Exposure duration: at least 48h at 37ºC in the dark.SELECTION AGENT (mutation assays):the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize an essential amino acid.NUMBER OF REPLICATIONS: 3 (except for one case, in which two plates were evaluated: Experiment 1, tester strain TA 1537, 100 μg/plate with S9).DETERMINATION OF CYTOTOXICITY- Method: Cytotoxicity could be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately <= 0.5 in relation to the solvent control.OTHER- Method of counting: the colonies were counted using a ProtoCOL counter. If precipitation of the test item precluded automatic counting the revertant colonies were counted by hand. In addition, tester strains with a low spontaneous mutation frequency like TA 1535 and TA 1537 were counted manually.
Evaluation criteria:
Evaluation of cytotoxicityCytotoxicity can be detected by a clearing rather disminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximatelly <= 0.5 in relation to the solvent control.Evaluation of MutagenicityThe Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).A test item is considered as mutagenic if:- a clear and dose-related increase in the number of revertants occurs and/or- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.A biologically relevant increase is described as follows:- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
2500 μg/plate and higher, experiment 2
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
5000 μg/plate, experiment 2
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS- Precipitation: No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)- Positive historical control data:Without S9TA 98: mean 444.5, SD 180.0, range 141-2213 (n= 1048)TA 100:mean 627.6, SD 255.3, range 132-1498 (n= 1195)TA 1535: mean 795.5, SD 323.7, range 34-1854 (n= 943)TA 1537: mean 92.0, SD 24.8, range 32-273 (n= 954)TA 102: mean 1801.0, SD 483.5, range 162-3321 (n=625)With S9TA 98: mean 2126.7, SD 679.0, range 113-3606 (n= 1032)TA 100:mean 1786.6, SD 494.6, range 169-3132 (n= 1194)TA 1535: mean 103.4, SD 64.9, range 20-1384 (n= 942)TA 1537: mean 221.8, SD 92.2, range 26-489 (n= 940)TA 102: mean 748.8, SD 176.3, range 137-1520 (n= 625)- Negative (solvent/vehicle) historical control data:Without S9TA 98: mean 23.1, SD 6.1, range 13-54 (n= 1035)TA 100: mean 89.2, SD 13.2, range 49-139 (n= 1190)TA 1535: mean 12.0, SD 5.8, range 4-39 (n= 944)TA 1537: mean 7.4, SD 2.6, range 2-35 (n= 943)TA 102: mean 251.4, SD 55.6, range 141-472 (n= 621)With S9TA 98: mean 29.9, SD 7.0, range 13-61 (n= 1033)TA 100:mean 98.3, SD 14.2, range 67-162 (n= 1196)TA 1535: mean 9.4, SD 3.7, range 4-32 (n= 943)TA 1537: mean 7.4, SD 2.8, range 3-36 (n= 941)TA 102: mean 317.7, SD 76.6, range 91-586 (n= 619)All criteria of validity regarding control plates were met.ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxic effects of the test item was observed in any of the five tested strain used up to the highest dose group evaluated (with and without metabolic activation) in Experiment 1 and 2.

Experiment 1: results for each strain and dose

Concentrations (μg/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella Thypimurium strains

 

 

TA 100

TA 102

TA1535

TA 1537

TA 98

 

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

31.6

Mean

93

98

261

276

21

15

6

5

25

26

MF

0.9

1.0

1.0

0.9

1.1

1.2

1.6

1.8

0.9

1.4

100

Mean

90

103

277

301

21

13

3

5

22

23

MF

0.9

1.1

1.1

1.0

1.1

1.0

0.7

0.8

0.8

1.3

316

Mean

92

100

298

301

19

17

6

5

19

24

MF

0.9

1.1

1.1

1.0

1.0

1.3

1.4

1.8

0.7

1.4

1000

Mean

89

102

281

284

21

14

7

5

28

25

MF

0.9

1.1

1.1

0.9

1.1

1.1

1.7

1.8

1.0

1.4

2500

Mean

97

102

281

304

20

17

10

3

27

27

MF

1.0

1.1

1.1

1.0

1.0

1.3

2.5

1.0

1.0

1.5

5000

Mean

124

113

275

315

22

16

7

5

24

23

MF

1.3

1.2

1.0

1.0

1.1

1.2

1.8

1.6

0.8

1.3

PC – I

Mean

665

-

646

-

847

-

92

-

374

-

MF

6.8

-

2.5

-

43.1

-

23.0

-

13.2

-

PC – II (2-AA)

Mean

-

1352

-

735

-

198

-

118

-

1461

MF

-

14.4

-

2.4

-

15.2

-

39.4

-

81.2

NC A. dest.

Mean

98

94

263

311

20

13

4

3

28

18

MF

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

Experiment 2: results for each strain and dose

Concentrations (μg/plate )

Mean values of revertants / Mutation factor (MF)

Salmonella Thypimurium strains

 

 

TA 100

TA 102

TA1535

TA 1537

TA 98

 

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

31.6

Mean

99

87

238

335

15

11

7

10

17

25

MF

1.4

0.9

1.1

0.9

1.6

0.8

1.0

1.3

0.6

1.1

100

Mean

94

101

259

345

15

13

8

10

20

33

MF

1.3

1.0

1.1

0.9

1.6

0.9

1.2

1.3

0.8

1.4

316

Mean

83

96

258

343

15

11

6

7

22

26

MF

1.1

1.0

1.1

0.9

1.6

0.8

0.9

1.0

0.8

1.2

1000

Mean

125

104

243

364

16

16

6

8

23

31

MF

1.7

1.1

1.1

1.0

1.8

1.1

1.0

1.0

0.9

1.4

2500

Mean

177

142

263

342

15

15

10

7

24

23

MF

2.4

1.5

1.2

1.2

1.6

1.1

1.6

1.0

0.9

1.0

5000

Mean

280

209

243

580

16

18

6

8

29

25

MF

3.8

2.2

1.1

1.6

1.7

1.3

1.0

1.1

1.1

1.1

PC – I

Mean

496

-

1987

-

632

-

90

-

400

-

MF

6.8

-

8.8

-

67.7

-

13.5

-

15.0

-

PC – II

Mean

-

1352

-

732

-

137

-

148

-

1084

MF

-

14.0

-

2.0

-

9.8

-

19.3

-

47.8

NC A. dest.

Mean

73

97

225

368

9

14

7

8

27

23

MF

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

Conclusions:
Under the experimental conditions reported, the test item was found to be mutagenic for the strain TA100.
Executive summary:

The aim of this test was to determine the ability of the test item to induce mutation, it was assessed using the bacterial reverse mutation test (Ames test). The test was performed following the OECD Guideline 471 with GLP on five Salmonella typhimurium strains. The performance of a pre-experiment for toxicity was not regarded as necessary. The test item dilutions were prepared in water for analysis for the main experiments. Two experiments were performed: experiment 1 with the plate incorporation method and the experiment 2 with the pre-incubation method. The following concentrations of the test item were prepared and used in these experiments: 31.6, 100, 316, 1000, 2500 and 5000 μL/plate. Additionally,positive and negative/solvent controls were included. After that, plates were incubated at 37ºC for at least 48h in the dark and after this period colonies were counted. No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation). No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I and II. No biologically relevant increases in revertant colony numbers were noted in tester strains TA 98, TA 1535, TA 1537 and TA 102. Biologically relevant increases of revertant colony numbers were observed in experiment II in tester strain TA 100 at concentrations of 2500 μg/plate and higher (without metabolic activation) and at a concentration of 5000 μg/plate (with metabolic activation). The threshold value of 2.0 was exceeded and a maximum mutation factor of 3.8 was reached at a concentration of 5000 μg/plate (without metabolic activation). In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item caused gene mutations by base pair changes in the genome of the tester strain TA 100.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro gene mutation study in bacteria: Key study: According to OECD 471 Guideline. GLP study. Five doses of the test item were evaluated on five S.thyphimurium strains. The test item was found to be mutagenic for the strain TA100 under the test conditions of pre-incubation.

In vitro gene mutation study in bacteria: Key study: According to OECD 471 Guideline. GLP study. Six doses of the test item were evaluated on five S.thyphimurium strains. Under the experimental conditions reported, the test item was found to be mutagenic for the strain TA100.

In vitro gene mutation study in bacteria: Key study: According to OECD 471 Guideline. GLP study. Six doses of the test item were evaluated on five S.thyphimurium strains. Under the experimental conditions reported, the test item was found to be mutagenic for strains TA100 and TA 102.

In vitro gene mutation study in bacteria: Key study: According to OECD 471 Guideline. GLP study. Six doses of the test item were evaluated on five S.thyphimurium strains. Under the experimental conditions reported, the test item was found to be mutagenic for the strain TA100.

In vitro cytogenicity / chromosome aberration study in mammalian cells: Experimental study on-going. According to OECD 473 Guideline. GLP study. Results are not available yet.

In vitro gene mutation study in mammalian cells: Experimental study on-giong. According to OECD 490 Guideline. GLP study. Results are not available yet.

Justification for selection of genetic toxicity endpoint:

Only in vitro gene mutation studies in bacteria are available.

Justification for classification or non-classification

Based on the positive Ames tests, the substance is classified as Mutagen Category 2 in accordance with CLP Regulation (EC) no 1272/2008.