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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: provided by the Sponsor
- Expiration date of the lot/batch: 06/11/2016.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: solvent water, maximal concentration in the solvent 94.5 g/l, stability in the solvent 48 hours

Method

Target gene:
Histidine-requiring gene in Salmonella typhimurium
Species / strainopen allclose all
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.
Controls
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.

Results and discussion

Test resultsopen allclose all
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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.