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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Identification: Bayscript Gelbkomponente
CAS-No.: 53523-90-3
Chemical Name: Tetralithium 5,5'-[vinylenebis[(3-sulphonato-4,1-phenylene)azo]]bis[3-methylsalicylate]
Empirical Formula: C30H20N4O12S2Li4
Molecular Mass: 720.4 g/mol
Appearance: Red-brown powder
Storage Conditions: At room temperature, moisture protected
Stability in Solvent: Stable for 4 and 24 hours at room temperature in the light in deion. Water (based on analytical study number PV29HC)
Expiry Date: 27 February 2017
Target gene:
histidine locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9 were used as the metabolic activation system
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate
with and without metabolic activation.

Since no toxic effects were observed, 5000 μg/plate were chosen as maximal concentration.
Vehicle / solvent:
Deionized water. The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionized water
True negative controls:
no
Positive controls:
yes
Remarks:
10 µg/plate, TA 1535, TA 100
Positive control substance:
sodium azide
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionized water
True negative controls:
no
Positive controls:
yes
Remarks:
10 µg/plate in strain TA 98. 50 µg/plater in strain TA 1537
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
2.0 µL/plate TA 102
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
2.5 µg/plate for all strains except TA 102 (10.0 µg/plate)
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
Precultures
The thawed bacterial suspension was transferred into 250 mL Erlenmeyer flasks containing 50 mL nutrient medium. A solution of 50 μL ampicillin (25 μg/mL) was added to the strains TA 98, TA 100, and TA 102. This nutrient medium contains per litre:
8 g Nutrient Broth 5 g NaCl
The bacterial cultures were incubated in a shaking water bath for 4 hours at 37° C. The optical density of the bacteria was determined by absorption measurement and the obtained values indicated that the bacteria were harvested at the late exponential or early stationary phase (10^8-10^9 cells/mL).

Pre-Experiment for Toxicity
To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I below (plate incorporation test).
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I, since the following criteria are met:
Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

Experimental Performance
For each strain and dose level, including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
Experiment I (Plate Incorporation)
100 μL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μL Bacteria suspension (cf. test system, pre-culture of the strains),
2000 μL Overlay agar
Experiment II (Pre-Incubation)
In the pre-incubation assay 100 μL test solution (solvent or reference mutagen solution (positive control)), 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.
In parallel to each test a sterile control of the test item was performed and documented in the raw data. Therefore, 100 μL of the stock solution, 500 μl S9 mix / S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates.

Data Recording
The colonies were counted using the Petri Viewer Mk2 with the software program Ames Study Manager.
Evaluation criteria:
Evaluation of Results
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
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
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
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
Species / strain:
S. typhimurium TA 100
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
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:
The test item Bayscript Gelbkomponente was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.
The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate
No precipitation of the test item occurred up to the highest investigated dose.
The plates incubated with the test item showed an increasing dense color from 333 to 5000 μg/plate, which had no impact on evaluation of the plates.
The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Bayscript Gelbkomponente at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct in-crease in induced revertant colonies.

            Summary of Experiment I

Study Name: 1747203

Study Code: Envigo 1747203

Experiment: 1747203 VV Plate

Date Plated: 11/03/2016

Assay Conditions:

Date Counted: 14/03/2016

 

Metabolic

Activation

Test

Group

Dose Level

(per plate)

 

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

TA 102

 

 

 

 

 

 

 

 

 

Without Activation

Deionised water

 

 

13 ± 3

7 ± 2

25 ± 5

160 ± 24

520 ± 8

Untreated

 

 

12 ± 1

10 ± 5

23 ± 1

160 ± 7

506 ± 6

Bayscript

3 µg

 

14 ± 2

9 ± 1

26 ± 6

170 ± 27

516 ± 12

Gelbkomponente

10 µg

 

15 ± 5

7 ± 2

20 ± 7

144 ± 19

539 ± 23

 

33 µg

 

13 ± 5

8 ± 1

21 ± 1

151 ± 16

511 ± 26

 

100 µg

 

14 ± 2

7 ± 2

24 ± 10

149 ± 17

495 ± 78

 

333 µg

 

13 ± 3D

11 ± 2D

24 ± 10D

168 ± 8D

519 ± 37D

 

1000 µg

 

12 ± 3D

8 ± 1D

25 ± 3D

142 ± 10D

539 ± 29D

 

2500 µg

 

13 ± 3D

7 ± 1D

20 ± 5D

152 ± 12D

530 ± 26D

 

5000 µg

 

10 ± 5D

7 ± 2D

21 ± 6D

143 ± 16D

503 ± 39D

NaN3

10 µg

 

1223 ± 29

 

 

2143 ± 132

 

4-NOPD

10 µg

 

 

 

390 ± 31

 

 

4-NOPD

50 µg

 

 

66 ± 12

 

 

 

MMS

2.0 µL

 

 

 

 

 

5562 ± 1178

 

 

 

 

 

 

 

 

 

With Activation

Deionised water

 

 

10 ± 1

11 ± 4

34 ± 2

153 ± 35

624 ± 42

Untreated

 

 

11 ± 4

16 ± 7

36 ± 7

119 ± 8

657 ± 12

Bayscript

3 µg

 

10 ± 3

10 ± 2

27 ± 2

131 ± 8

762 ± 28

Gelbkomponente

10 µg

 

9 ± 3

11 ± 4

27 ± 10

134 ± 26

754 ± 55

 

33 µg

 

8 ± 1

7 ± 1

26 ± 6

142 ± 5

713 ± 63

 

100 µg

 

13 ± 6

10 ± 4

32 ± 3

128 ± 5

733 ± 130

 

333 µg

 

14 ± 2D

10 ± 2D

34 ± 4D

132 ± 13D

734 ± 15D

 

1000 µg

 

11 ± 4D

6 ± 1D

26 ± 7D

122 ± 15D

692 ± 48D

 

2500 µg

 

11 ± 5D

10 ± 3D

26 ± 4D

129 ± 8D

675 ± 82D

 

5000 µg

 

14 ± 2D

10 ± 5D

37 ± 1D

143 ± 7D

666 ± 103D

2-AA

2.5 µg

 

426 ± 17

112 ± 8

4516 ± 613

2495 ± 184

 

2-AA

10.0 µg

 

 

 

 

 

1715 ± 215

 

 

 

 

 

 

 

 

 

 

Key to Positive Controls

Key to Plate Postfix Codes

 

 

NaN3

2-AA

MMS

4-NOPD

sodium azide

2-aminoanthracene

methyl methane sulfonate

4-nitro-o-phenylene-diamine

D

Densely coloured plate

Summary of Experiment II

Study Name: 1747203

Study Code: Envigo 1747203

Experiment: 1747203 HV2 Pre

Date Plated: 24/03/2016

Assay Conditions:

Date Counted: 30/03/2016

 

Metabolic

Activation

Test

Group

Dose Level

(per plate)

 

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

TA 102

 

 

 

 

 

 

 

 

 

Without Activation

Deionised water

 

 

9 ± 2

8 ± 1

21 ± 2

192 ± 7

451 ± 31

Untreated

 

 

12 ± 2

8 ± 2

24 ± 3

196 ± 14

471 ± 30

Bayscript

33 µg

 

9 ± 3

8 ± 2

22 ± 6

215 ± 22

453 ± 25

Gelbkomponente

100 µg

 

8 ± 3

8 ± 1

23 ± 4

177 ± 17

443 ± 11

 

333 µg

 

10 ± 3D

8 ± 2D

24 ± 3D

194 ± 9D

451 ± 70D

 

1000 µg

 

8 ± 2D

7 ± 2D

23 ± 3D

201 ± 4D

453 ± 33D

 

2500 µg

 

9 ± 2D

7 ± 1D

19 ± 4D

197 ± 12D

395 ± 36D

 

5000 µg

 

10 ± 2D

9 ± 4D

25 ± 1D

207 ± 21D

351 ± 62D

NaN3

10 µg

 

1057 ± 47

 

 

1977 ± 31

 

4-NOPD

10 µg

 

 

 

408 ± 30

 

 

4-NOPD

50 µg

 

 

109 ± 15

 

 

 

MMS

2.0 µL

 

 

 

 

 

5449 ± 739

 

 

 

 

 

 

 

 

 

With Activation

Deionised water

 

 

11 ± 2

10 ± 5

29 ± 2

199 ± 27

603 ± 18

Untreated

 

 

7 ± 1

11 ± 4

38 ± 2

180 ± 18

643 ± 47

Bayscript

33 µg

 

9 ± 3

10 ± 5

30 ± 1

185 ± 23

648 ± 49

Gelbkomponente

100 µg

 

10 ± 3

9 ± 2

35 ± 8

198 ± 10

585 ± 5

 

333 µg

 

12 ± 4D

11 ± 1D

34 ± 6D

202 ± 15D

563 ± 118D

 

1000 µg

 

11 ± 4D

11 ± 2D

25 ± 7D

202 ± 15D

583 ± 27D

 

2500 µg

 

14 ± 2D

10 ± 2D

26 ± 3D

211 ± 11D

551 ± 31D

 

5000 µg

 

15 ± 2D

9 ± 3D

28 ± 3D

181 ± 8D

449 ± 7D

2-AA

2.5 µg

 

417 ± 45

106 ± 13

4377 ± 111

2370 ± 210

 

2-AA

10.0 µg

 

 

 

 

 

1872 ± 278

 

 

 

 

 

 

 

 

 

 

Key to Positive Controls

Key to Plate Postfix Codes

 

 

NaN3

2-AA

MMS

4-NOPD

sodium azide

2-aminoanthracene

methyl methane sulfonate

4-nitro-o-phenylene-diamine

D

Densely coloured plate

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Executive summary:

This study was performed to investigate the potential of Bayscript Gelbkomponente to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item, dissolved in deionized water, was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate

No precipitation of the test item occurred up to the highest investigated dose.

The plates incubated with the test item showed an increasing dense color from 333 to 5000 μg/plate, which had no impact on evaluation of the plates.

The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Bayscript Gelbkomponente at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Conclusion

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, Bayscript Gelbkomponente is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan-April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro mammalian forward mutation assay
Specific details on test material used for the study:
Appearance: Red-brown powder
Storage Conditions: At room temperature, moisture protected
Expiry Date: 27 February 2017
Stability in Solvent: Stable over 4 and 24 hours at room temperature in the light in deion. Water (based on analytical study number PV29HC)

A correction for purity was made
Target gene:
HPRT (hypoxanthine-guanine phosphoribosyl transferase) gene locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.

Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of Envigo CRS GmbH allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants may be reduced by treatment with HAT-medium. Each master cell stock is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
Thawed stock cultures were propagated at 37 °C in 75 cm2 plastic flasks. About 2-3×10^6 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%). The cells were sub-cultured once or twice weekly.
All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.

For seeding of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 μg/mL), 10% FBS, and amphotericin B (1 %). During treatment no FBS was added to the medium. For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9 was used as metabolic activation system.
Test concentrations with justification for top dose:
Pre-experiment toxicity test
With and without S9-mix: 18.8, 37.6, 75.1, 150.3, 300.5, 601.0, 1202.0 and 2404.0 µg/ml
The highest concentration (2404 µg/mL equal to approximately 2000 µg/mL of the pure substance) was chosen with respect to the current OECD Guideline 476 and the purity of the test item

In the pre-experiment no relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was observed up to the highest concentration with and without metabolic activation.
The test medium was checked for precipitation or phase separation at the beginning and at the end of treatment (4 hours) prior to removal to the test item. No precipitation or phase separation occurred after 4 hours treatment with and without metabolic activation.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.

Main experiment:
With and without S9-mix:75.1, 150.3, 300.5, 601.0, 1202.0 and 2404.0 µg/ml
The dose range of the main experiment was set according to data generated in the pre-experiment. The individual concentrations were spaced by a factor of 2.0. To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations.
Vehicle / solvent:
The vehicle was deionised water. The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Remarks:
Final concentration: 300 μg/mL = 2.4 mM
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Remarks:
Final concentration: 2.3 μg/mL = 8.9 μM
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
With metabolic activation
Details on test system and experimental conditions:
Experimental Design and Study Conduct
Pre-Test on Toxicity
A pre-test was performed in order to determine the concentration range for the mutagenicity experiment. The general culture conditions and experimental conditions in this pre-test were the same as described for the mutagenicity experiment below. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).

Experimental Performance
The experiment was performed with a treatment time of 4 hours with and without metabolic activation. Two parallel cultures were used throughout the assay.

Seeding
Two to four days after sub-cultivation stock cultures were trypsinized at 37 °C for approximately 5 to 10 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10% FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2% in saline.
Prior to the trypsin treatment the cells were rinsed with PBS. Approximately 0.7 to 1.2 x 10^7 were seeded in plastic flasks. The cells were grown for 24 hours prior to treatment.

Treatment
After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 µl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. 4 hours after treatment, this medium was replaced with complete medium following two washing steps with "saline G".
Immediately after the end of treatment the cells were trypsinised as described above and sub-cultivated. At least 2.0 x 10^6 cells per experimental point (concentration series plus controls) were subcultured in 175 cm² flasks containing 30 mL medium.
Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (cloning efficiency I) as measure of test item induced cytotoxicity. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2.
The colonies used to determine the cloning efficiency I were fixed and stained 6 to 8 days after treatment as described below.
Three or four days after first sub-cultivation approximately 2.0 x 10^6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium.
Following the expression time of 7 days five 75 cm² cell culture flasks were seeded with about 4 to 5 x 10^5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability (cloning efficiency II).
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 for about 8 days. The colonies were stained with 10% methylene blue in 0.01% KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.
Evaluation criteria:
HPRT catalyzes the conversion of the nontoxic 6-TG (6-thioguanine) to its toxic ribophosphorylated derivative. Therefore, cells deficient in HPRT due to a forward mutation are resistant to 6-TG. These cells are able to proliferate in the presence of 6-TG whereas the non-mutated cells die.

A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.

A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares).
Statistics:
A linear regression analysis (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The test item Bayscript Gelbkomponente was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The treatment period was 4 hours with and without metabolic activation.

A minor reduction of the relative adjusted cloning efficiency below 50% in both parallel cultures was noted at 150.3 µg/mL. This effect however, was not judged as real cytotoxic effect as no reduction of the relative adjusted cloning efficiency was observed at any other of the higher concentrations or at the same concentration of the pre-experiment.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined with any of the experimental groups.
No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiment up to the maximum concentration.
The 95% confidence interval was not exceeded at any concentration of the test item.
In the main experiment with and without S9 mix the range of the solvent controls was from 14.3 up to 26.4 mutants per 10^6 cells; the range of the groups treated with the test item was from 8.5 up to 23.5 mutants per 10^6 cells.
EMS (300 µg/mL) and DMBA (2.3 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusions:
In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, Bayscript Gelbkomponente is considered to be non-mutagenic in this HPRT assay.

Executive summary:

Summary

The study was performed to investigate the potential of Bayscript Gelbkomponente to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The treatment period was 4 hours with and without metabolic activation.

The maximum concentration of the pre-test on toxicity and and the main experiment was 2404 µg/mL equal to approximately 2000 µg/mL of the pure substance as requested by the current OECD Guideline 476.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Conclusion

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, Bayscript Gelbkomponente is considered to be non-mutagenic in this HPRT assay.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April-June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
Expiry / Retest Date: 27 February 2017
Storage Conditions: At room temperature, moisture protected
Appearance: red-brown powder
Stability in Solvent: Stable over 4 and 24 hours at room temperature in the light in deion. water (based on analytical studies, Envigo reference number PV29HC)
Correction for purity was made.

Target gene:
Human lymphocytes - micronucleus
Species / strain / cell type:
lymphocytes: Human
Details on mammalian cell type (if applicable):
Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours. The cell harvest time point was approximately 2 – 2.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 Mix
Test concentrations with justification for top dose:
Dose selection will be performed according to the current OECD Guideline for the in vitro micronucleus test. The highest test item concentration will be 10 mM, 2 mg/mL or, 2 µL/mL, whichever is the lowest. When the test item is not of defined composition (e.g. UVCBs or environmental extracts) the top concentration may need to be higher (e.g. 5 mg/mL) in the absence of sufficient cytotoxicity. At least three test item concentrations should be evaluated for cytogenetic damage.
For full doses see table in section 'Any additional information on materials and methods'
Vehicle / solvent:
Stock formulations of the test item and serial dilutions were made in deionised water. The final concentration of deionised water in the culture medium was 10%. The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
culture medium with 10.0 % deionised water (local tap water deionized at Envigo CRS GmbH)
True negative controls:
no
Positive controls:
yes
Remarks:
Dissolved in deionised water. Concentration: 1.0 µg/mL
Positive control substance:
mitomycin C
Remarks:
Without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
culture medium with 10.0 % deionised water (local tap water deionized at Envigo CRS GmbH)
True negative controls:
no
Positive controls:
yes
Remarks:
Dissolved in deionised water. Concentration: 50.0 ng/mL
Positive control substance:
other: Demecolcin
Remarks:
Without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
culture medium with 10.0 % deionised water (local tap water deionized at Envigo CRS GmbH)
True negative controls:
no
Positive controls:
yes
Remarks:
Dissolved in Saline (0.9 % NaCl [w/v]). Concentration: 17.5 µg/mL
Positive control substance:
cyclophosphamide
Remarks:
With metabolic acitvation
Details on test system and experimental conditions:
Test System and Supporting Information:
Reason for the choice of human lymphocytes:
Human lymphocytes are the most common cells in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level.

Cell cultures:
Blood samples were drawn from healthy non-smoking donors not receiving medication. For this study, blood was collected from a female donor (33 years old) for Experiment I and from a female donor (27 years old) for Experiment II. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours. The cell harvest time point was approximately 2 – 2.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.

Culture conditions:
Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL), the mitogen PHA (3 µg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

Mammalian Microsomal Fraction S9 Mix:
Due to the limited capacity for metabolic activation of potential mutagens in in vitro methods an exogenous metabolic activation system was used.
Phenobarbital/β-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared and stored according to the currently valid version of the Envigo SOP for rat liver S9 preparation. Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
The protein concentration of the S9 preparation used for this study was 27.2 mg/mL (Lot no. 240915).

Experimental Design and Study Conduct:
Dose Selection:
Dose selection will be performed according to the current OECD Guideline for the in vitro micronucleus test. The highest test item concentration will be 10 mM, 2 mg/mL or, 2 µL/mL, whichever is the lowest. When the test item is not of defined composition (e.g. UVCBs or environmental extracts) the top concentration may need to be higher (e.g. 5 mg/mL) in the absence of sufficient cytotoxicity. At least three test item concentrations should be evaluated for cytogenetic damage.
With regard to the purity (83.2%) of the test item, 2404 µg/mL were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 15.62 to 2404 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, no precipitation of the test item was observed. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
Using a reduced Cytokinesis-block proliferation index (CBPI) as an indicator for toxicity, no cytotoxic effects were observed in Experiment I after 4 hours treatment in the absence and presence of S9 mix. Therefore, 2404 µg/mL were chosen as top treatment concentration for Experiment II.
The cytogenetic evaluation of concentrations in Experiment II (without S9 mix) higher than indicated in Table 1 was impossible due to strong test item-induced toxic effects (low cell numbers).

Pre-experiment:
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of reduction in the CBPI in comparison with the controls (% cytostasis) by counting 500 cells per culture. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay.
The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 40 hrs after start of the exposure.

Cytogenetic Experiment::
Pulse exposure:
About 48 hrs after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 µL S9 mix per mL culture medium was added. After 4 hrs the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were resuspended in and washed with "saline G" (pH 7.2, containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose • H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4). The washing procedure was repeated once as described. The cells were resuspended in complete culture medium with 10 % FBS (v/v) and cultured for a 16-hour recovery period. After this period Cytochalasin B (4 µg/mL) was added and the cells were cultured another approximately 20 hours until preparation.

Continuous exposure (without S9 mix):
About 48 hrs after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with complete medium (with 10 % FBS) containing the test item. After 20 hours the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were re-suspended in and washed with "saline G". The washing procedure was repeated once as described. After washing the cells were re-suspended in complete culture medium containing 10 % FBS (v/v). Cytochalasin B (4 µg/mL) was added and the cells were cultured another approximately 20 hours until preparation.

Preparation of cells:
The cultures were harvested by centrifugation 40 hrs after beginning of treatment. The cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were re-suspended in approximately 5 mL saline G and spun down once again by centrifugation for 5 minutes. Then the cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37 °C for 20 minutes. 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa.
Evaluation criteria:
The micronucleus assay will be considered acceptable if it meets the following criteria:
 The concurrent solvent control will normally be within the laboratory historical solvent control data range.
 The concurrent positive controls should induce responses that are compatible with the laboratory historical positive control data and produce a statistically significant increase.
 Cell proliferation criteria in the solvent control are considered to be acceptable.
 All experimental conditions described in section ‘Experimental performance’ were tested unless one exposure condition resulted in a clearly positive result.
 The quality of the slides must allow the evaluation of an adequate number of cells and concentrations.
 The criteria for the selection of top concentration are consistent with those described in section ‘Dose selection’.
Statistics:
Statistical significance will be confirmed by the Chi square test (α < 0.05), using the validated R Script CHI2.Rnw for those values that indicate an increase in the number of cells with micronuclei compared to the concurrent solvent control. Other statistical methods may be used if appropriate.
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The test item Bayscript Gelbkomponente, dissolved in deionised water, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analyzed. At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis.
The highest treatment concentration in this study, 2404 µg/mL was chosen with regard to the purity of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.
No precipitation of the test item in the culture medium was observed. No relevant influence on osmolarity or pH was observed.
In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II no cytotoxicity was observed up to the highest evaluated concentration. The highest concentration, showing clear cytotoxic effects, was not evaluable for cytogenetic damage.
In the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item.
In Experiment I in the absence of S9 mix, however, the value of 1.40 % micronucleated cells after treatment with 785 µg/mL was outside the range of the laboratory historical solvent control data (95 % control limit: 0.02 – 1.15 %). Since the increase was not statistically significant, the effect has to be considered as biologically irrelevant.
In the presence of S9 mix the number of micronucleated cells in the solvent control (1.23 %) slightly exceeded the historical control range (95 % control limit: 0.08 – 1.20 %). This is considered as biological variation and has no influence on the validity of the test system.
In both experiments, either Demecolcin (50.0 ng/mL), MMC (1.0 µg/mL) or CPA (17.5 µg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

Summary of results

Exp.

Preparation

Test item

Proliferation

Cytostasis

Micronucleated

 

interval

concentration

index

in %*

cells

 

 

in µg/mL

CBPI

 

in %**

Exposure period 4 hrs without S9 mix

I

40 hrs

Solvent control1

1.99

 

1.10

 

 

Positive control2

1.98

1.2

 10.35S

 

 

785#

2.02

n.c.

1.40

 

 

1374#

2.00

n.c.

1.05

 

 

2404

1.95

4.1

1.15

Exposure period 20 hrs without S9 mix

II

40 hrs

Solvent control1

1.76

 

0.65

 

 

Positive control3

1.51

33.1

 3.10S

 

 

449

1.58

23.6

0.50

 

 

785

1.54

29.2

0.55

 

 

1374

1.55

27.4

0.75

Exposure period 4 hrs with S9 mix

I

40 hrs

Solvent control1#

2.10

 

1.23

 

 

Positive control4

1.78

29.4

 8.70S

 

 

785#

2.07

3.1

1.18

 

 

1374#

2.03

6.3

1.00

 

 

2404#

2.04

6.2

1.15

*      For the positive control groups and the test item treatment groups the values are related to the solvent controls

**    The number of micronucleated cells was determined in a sample of 2000 binucleated cells

#       The number of micronucleated cells was determined in a sample of 4000 binucleated cells

S       The number of micronucleated cells is statistically significantly higher than corresponding control values

n.c.  Not calculated as the CBPI is equal or higher than the solvent control value

1       Deion. water   10.0 % (v/v)
2
           MMC                 1.0 µg/mL

3           Demecolcin    50.0 ng/mL

4           CPA                 17.5 µg/mL

 

Conclusions:
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, Bayscript Gelbkomponente is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required or evaluable concentration.
Executive summary:

The test item Bayscript Gelbkomponente, dissolved in deionised water, was assessed for its potential to induce micronuclei in human lymphocytesin vitroin two independent experiments. The following study design was performed:

 

Without S9 mix

With S9 mix

 

Exp. I

Exp. II

Exp. I

Stimulation period

 48 hrs

48 hrs

 48 hrs

Exposure period

 4 hrs

20 hrs

 4 hrs

Recovery

16 hrs

¾

16 hrs

Cytochalasin B exposure

20 hrs

20 hrs

20 hrs

Total culture period

88 hrs

88 hrs

88 hrs

In each experimental group two parallel cultures were analyzed. Per culture at least1000 binucleated cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (2404 µg/mL of the test item) was chosen with regard to the purity (83.2%) of the test item and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487.

In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II no cytotoxicity was observed up to the highest evaluated concentration. However, concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage.

In the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item.   
In Experiment I in the absence of S9 mix, however, the value of 1.40 % micronucleated cells after treatment with 785 µg/mL was outside the range of the laboratory historical solvent control data (95 % control limit: 0.02 – 1.15 %). Since the increase was not statistically significant, the effect has to be considered as biologically irrelevant.
                       
In the presence of S9 mix the number of micronucleated cells in the solvent control (1.23 %) slightly exceeded the historical control range (95 % control limit: 0.08 – 1.20 %). This is considered as biological variation and has no influence on the validity of the test system.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.


Conclusion

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by thein vitromicronucleus test in human lymphocytes.

Therefore, Bayscript Gelbkomponente is considered to benon-mutagenic in thisin vitromicronucleustest, when tested up to the highest required or evaluable concentration.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In the Ames-Test according to OECD TG 407 the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Furthermore, it did not induce gene mutations at the HPRT locus in V79 cells when tested in a study according to OECD TG 476. The test item did also not induce micronuclei as determined in an in vitro micronucleus test in human lymphocytes according to OECD TG 487.

Overall, it is not considered that the substance has a genotoxic potential.

Justification for classification or non-classification

No classification concluded for Genetic Toxicity according to Regulation (EC) No 1272/2008, Annex I.