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Genetic toxicity in vitro

Description of key information

In vitro reverse gene mutation assay in bacteria: Negative; OECD 471; Schulz, M. (2018)

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 July - 27 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordance with OECD guidelines and GLP. All guideline validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix derived from rat liver.
Test concentrations with justification for top dose:
Pre-Experiment I/Ia

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 following concentrations were tested in the pre- experiment/experiment I; 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Toxicity of the test item results in a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn.
Since the acceptance criteria were not met in the pre-experiment in strains TA 98 and TA 100 without S9 mix, this part of experiment I was repeated as plate-incorporation assay with the following concentrations (experiment Ia); 0.03; 0.1; 0.3; 1; 3; 10; 33; and 100 μg/plate

Experiment II

TA 98 and TA 100 (-S9 mix): 0.03, 0.1, 0.1, 1, 3, 10, 33 and 100 µg/plate
TA 98 and TA 100 (+S9 mix): 0.3, 1, 3, 10, 33, 100, 333 and 1000 µg/plate
TA 1537 and WP2uvra (+/- S9 mix): 0.3, 1, 3, 10, 33, 100, 333 and 1000 µg/plate

Since the lower limit of the positive controls historical control data were not reached in strain TA98 without S9 mix and the expected toxicity was not observed in strains TA 1535 and WP2 uvrA with S9 mix, this part of experiment II was repeated as pre-incubation assay with the following concentrations (experiment IIa)

Experiment IIa

TA 98 (-S9 mix): 0.03, 0.1, 0.3, 1, 3, 10, 33 and 100 µg/plate
TA 1535 and WP2 uvra (+S9 mix): 3, 10, 33, 100, 33, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Test item = ethanol (soluble); positive controls = DMSO
- Justification for choice of solvent/vehicle: Solvents chosen because of there solubility properties and relative nontoxicity to the bacteria (Maron et al.; 1981).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 4-NOPD (-S9 mix): 10 μg/plate in strain TA 98, 50 μg/plate in strain TA 1537 and 2-aminoanthracene, 2-AA (+S9 mix): 2.5 μg/plate TA 1535, TA 1537, TA 98, TA 100 & 10.0 μg/plate in WP2 uvrA
Details on test system and experimental conditions:
For each strain and dose level, including the controls, three plates were used.

Experiment I (Plate Incorporation)

The following materials were mixed in a test tube and poured onto the selective agar plates:
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. 3.4.3 Precultures), 2000 μL Overlay agar

Experiment II / IIa (Pre-Incubation)

The following materials were mixed in a test tube and incubated at 37°C for 60 minutes;
50 μL Test solution at each dose level (solvent control),
100 μL 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. 3.4.3 Precultures).

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 (experiment I) or 50 μL (experiment II) 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.
Evaluation criteria:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
• regular background growth in the negative and solvent control;
• the spontaneous reversion rates in the negative and solvent control are in the range of historical data;
• the positive control substances should produce an increase above the threshold of twofold (strains TA 98, TA 100, and WP2 uvrA) or threefold (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control;
• a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
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:
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:
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 and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not investigated
- Effects of osmolality: Not investigated
- Evaporation from medium: Not investigated
- Precipitation: In the experiments I, II, and IIa precipitation of the test item occurred in the overlay agar in the test tubes from 1000 μg/plate up the highest investigated dose. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 μg/plate up to the highest investigated dose in the experiments I, II, and II a. The undissolved particles had no influence on the data recording. Based on a lower top dose (100 μg/plate) no precipitation of the test item occurred neither in the test tubes nor on the incubated agar plates in experiment Ia and II in strain TA 98 and TA 100 without S9 mix and in experiment IIa in strain TA 98 without S9 mix.
- Definition of acceptable cells for analysis:

RANGE-FINDING/SCREENING STUDIES: Experiment 1 and 1a reported.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: N/A

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: N/A
- Indication whether binucleate or mononucleate where appropriate: N/A

HISTORICAL CONTROL DATA
- The laboratory ́s historical control data from November 2014 until November 2016 representing approx. 600 experiments (WP2 uvrA the historical data are based on approx. 350 experiments). Please refer to study report Annex 1.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Visual inspection of bacterial lawn. A reduction in bacterial lawn indicating a cytotoxic effect.
- Other observations when applicable: N/A

Table1       Summary results of Experiment I (plate incorporation method)

 

Metabolic activation

Test group

Dose level

(µg/plate)

Revertant colony counts (mean ± SD)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Without

Solvent control

-

10 ± 3

9 ± 2

33 ± 3

181 ± 6

44 ± 1

Negative control

-

15 ± 4

10 ± 3

25 ± 7

175 ± 8

40 ± 7

Test item

3

10 ± 0

9 ± 2

29 ± 4

114 ± 7

40 ± 12

10

9 ± 3

9 ± 2

30 ± 3

68 ± 9 R

40 ± 2

33

6 ± 3

9 ± 2 R

17 ± 3 MR

21 ± 7 MR

42 ± 3

100

5 ± 1 MR

1 ± 2 MR

6 ± 2 MR

4 ± 1 MR

40 ± 6

333

2 ± 1 MR

0 ± 1 MR

0 ± 0 MR

1 ± 1 R

26 ± 1 R

1000

0 ± 1 PMR

0 ± 0 PMR

0 ± 0 PMR

0 ± 0 PR

8 ± 2 PMR

2500

0 ± 1 PMR

0 ± 0 PMR

0 ± 0 PMR

0 ± 0 PMR

4 ± 2 PMR

5000

0 ± 0 PMR

0 ± 0 PMR

0 ± 0 PMR

0 ± 0 PMR

2 ± 0 PMR

NaN3

10

1064 ± 46

-

-

1632 ± 118

-

4-NOPD

10

-

-

434 ± 10

-

-

4-NOPD

50

-

70 ± 7

-

-

-

MMS

2.0

-

-

-

-

900 ± 79

With

Solvent control

-

14 ± 5

10 ± 3

43 ± 2

182 ± 14

51 ± 9

Negative control

-

18 ± 3

13 ± 1

48 ± 7

170 ± 12

59 ± 5

Test item

3

14 ± 3

15 ± 2

52 ± 6

172 ± 32

54 ± 3

10

13 ± 3

12 ± 5

49 ± 12

170 ± 9

64 ± 2

33

15 ± 5

11 ± 3

43 ± 7

156 ± 14

41 ± 6

100

11 ± 1

13 ± 3

42 ± 16

125 ± 14

46 ± 5

333

13 ± 3 R

13 ± 3 R

6 ± 2 MR

4 ± 1 MR

54 ± 6

1000

3 ± 1 PMR

2 ± 1 PMR

0 ± 0 PMR

1 ± 1 PMR

14 ± PMR

2500

0 ± 1 PMR

0 ± 0 PMR

0 ± 0 PMR

0 ± 0 PMR

4 ± 1 PMR

5000

0 ± 0 PMR

0 ± 0 PMR

0 ± 0 PMR

0 ± 0 PMR

2 ± 1 PMR

2-AA

2.5

184 ± 17

312 ± 51

1919 ± 425

2589 ± 218

-

2-AA

10

-

-

-

-

186 ± 24

 

Table2       Summary results of Experiment Ia (plate incorporation method repeat)

 

Metabolic activation

Test group

Dose level

(µg/plate)

Revertant colony counts (mean ± SD)

TA 98

TA 100

Without

Solvent control

-

34 ± 6

152 ± 17

Negative control

-

29 ± 8

163 ± 18

Test item

0.03

28 ± 4

156 ± 17

0.1

32 ± 1

153 ± 9

0.3

29 ± 7

139 ± 3

1

28 ± 7

125 ± 6

3

30 ± 8

125 ± 6

10

28 ± 3

109 ± 14

33

13 ± 4 MR

77 ± 13

100

2 ± 1 MR

18 ± 5 MR

4-NOPD

10

397 ± 27

2 ± 1 MR

NaN3

10

-

1681 ± 119

 

Table3       Summary results of Experiment II (pre-incubation method)

 

Metabolic activation

Test group

Dose level

(µg/plate)

Revertant colony counts (mean ± SD)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Without

Solvent control

-

11 ± 3

9 ± 2

32 ± 5

189 ± 14

46 ± 13

Negative control

-

11 ± 2

9 ± 4

39 ± 3

209 ± 7

45 ± 4

Test item

0.03

-

-

22 ± 6

202 ± 19

-

0.1

-

-

36 ± 10

192 ± 4

-

0.3

10 ± 2

10 ± 2

31 ± 5

193 ± 17

46 ± 10

1

11 ± 5

12 ± 4

31 ± 8

183 ± 10

42 ± 8

3

10 ± 1

12 ± 4

25 ± 6

63 ± 10

29 ± 6

10

9 ± 2

10 ± 3

27 ± 2

61 ± 20

48 ± 3

33

5 ± 1

9 ± 4

4 ± 1

23 ± 1

34 ± 5

100

1 ± 1

4 ± 2

0 ± 1

1 ± 1

23 ± 4

333

1 ± 1

1 ± 1 R

-

-

27 ± 8 R

1000

0 ± 0 P

1 ± 1 PMR

-

-

19 ± 3 PMR

NaN3

10

962 ± 41

-

-

1529 ± 122

-

4-NOPD

10

-

-

25 ± 7

-

-

4-NOPD

50

-

90 ± 4

-

-

-

MMS

2

-

-

-

-

532 ± 39

With

Solvent control

-

17 ± 6

17 ± 3

48 ± 3

203 ± 8

54 ± 8

Negative control

-

15 ± 1

15 ± 3

44 ± 3

208 ± 6

58 ± 11

Test item

0.3

16 ± 2

21 ± 5

54 ± 7

200 ± 11

47 ± 7

1

12 ± 3

13 ± 1

59 ± 11

202 ± 24

49 ± 6

3

18 ± 2

14 ± 5

50 ± 7

202 ± 18

50 ± 6

10

10 ± 1

18 ± 3

48 ± 6

228 ± 7

61 ± 3

33

11 ± 4

20 ± 6

53 ± 8

205 ± 3

52 ± 9

100

12 ± 6

20 ± 3 R

52 ± 11

161 ± 17

61 ± 6

333

15 ± 5

14 ± 4 MR

19 ± 5 MR

54 ± 5 MR

49 ± 3

1000

14 ± 3 PM

7 ± 2 PMR

1 ± 1 PMR

7 ± 3 PMR

36 ± 7 PMR

2-AA

2.5

200 ±20

295 ± 16

2829 ± 575

3258 ± 26

-

2-AA

10

-

-

-

-

228 ± 2

 

Table4       Summary results of Experiment IIa (pre-incubation method repeat)

 

Metabolic activation

Test group

Dose level

(µg/plate)

Revertant colony counts (mean ± SD)

TA 1535

TA 98

WP2 uvrA

Without

Solvent control

-

-

40 ± 3

-

Negative control

-

-

27 ± 11

-

Test item

0.03

-

31 ± 7

-

0.1

-

32 ± 6

-

0.3

-

29 ± 3

-

1

-

33 ± 10

-

3

-

28 ± 7

-

10

-

21 ± 6

-

33

-

1 ± 1

-

100

-

0 ± 0

-

4-NOPD

10

-

526 ± 18

-

With

Solvent control

-

14 ± 4

-

43 ± 2

Negative control

-

16 ± 4

-

52 ± 10

Test item

3

13 ± 6

-

42 ± 4

10

13 ± 5

-

48 ± 4

33

15 ± 2

-

39 ± 2

100

13 ± 3

-

51 ± 11

333

9 ± 2

-

41 ± 11 R

1000

5 ± 2 PM

-

15 ± 4 PMR

2500

4 ± 1 PM

-

8 ± 2 PMR

5000

2 ± 1 PM

-

1 ± 1 PMR

2-AA

2.5

202 ± 23

-

-

2-AA

10

-

-

208 ± 21

NaN3: sodium azide

2-AA: 2-aminoanthracene

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

MMS: methyl methane sulfonate

R: reduced background growth

M: manual count

P: precipitate

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:

OECD 471 (2018) - In a reverse gene mutation assay in bacteria using strains of S. typhimurium (TA1535, TA1537, TA98, TA100) and Escherichia coli (WP2 uvrA), the test item was assayed for mutagenic potential both in the absence and in the presence of metabolic activation by an induced rat liver post-mitochondrial fraction (S-9), in two independent experiments with the inclusion of pre-incubation step. The test item treatments in this study were performed using formulations prepared in ethanol.

 

Experiment I/Ia (repeat) treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of the test item at 0.03-5000 μg/plate, plus vehicle, negative and positive controls. Following these treatments, evidence of toxicity was observed at 1000 μg/plate (respectively, 100 µg/plate in strains TA98 and TA100 without S9-mix).

 

Experiment II/IIa (repeat) treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 1000 μg/plate was retained for all strains (Experiment IIa repeat conducted up to 5000 µg/plate for strain TA1535 and WP2 uvrA with S9-mix in the absence of expected toxicity in Experiment II at 1000 µg/plate).

 

Following these treatments, evidence of toxicity was observed in all strains in the absence of S9 and in the presence of S9 mix.

 

Precipitation was observed in the overlying agar in test tubes >1000 µg/plate doses. Undissolved particles had no influence on the data recording.

 

Vehicle, negative and positive control treatments were included for all strains in all experiments. The mean numbers of revertant colonies fell within acceptable ranges for vehicle control treatments, and were elevated by positive control treatments.

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with AD-633 at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix).

 

It was concluded that under the condition of this study, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains tested. These conditions included treatments at concentrations up to 5000 μg/plate (the maximum recommended concentration according to current regulatory guidelines), in the absence and in the presence of a rat liver metabolic activation system (S-9).

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Not applicable, no adverse effects observed.

Additional information

Genetic toxicity - in vitro

OECD 471 (2018)- In a reverse gene mutation assay in bacteria using strains of S. typhimurium (TA1535, TA1537, TA98, TA100) and Escherichia coli (WP2 uvrA), the test item was assayed for mutagenic potential both in the absence and in the presence of metabolic activation by an induced rat liver post-mitochondrial fraction (S-9), in two independent experiments with the inclusion of pre-incubation step. The test item treatments in this study were performed using formulations prepared in ethanol.

 

Experiment I/Ia (repeat) treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of the test item at 0.03-5000 μg/plate, plus vehicle, negative and positive controls. Following these treatments, evidence of toxicity was observed at 1000 μg/plate (respectively, 100 µg/plate in strains TA98 and TA100 without S9-mix).

 

Experiment II/IIa (repeat) treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 1000 μg/plate was retained for all strains (Experiment IIa repeat conducted up to 5000 µg/plate for strain TA1535 and WP2 uvrA with S9-mix in the absence of expected toxicity in Experiment II at 1000 µg/plate).

 

Following these treatments, evidence of toxicity was observed in all strains in the absence of S9 and in the presence of S9 mix.

 

Precipitation was observed in the overlying agar in test tubes >1000 µg/plate doses. Undissolved particles had no influence on the data recording.

 

Vehicle, negative and positive control treatments were included for all strains in all experiments. The mean numbers of revertant colonies fell within acceptable ranges for vehicle control treatments, and were elevated by positive control treatments.

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with AD-633 at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix).

 

It was concluded that under the condition of this study, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains tested. These conditions included treatments at concentrations up to 5000 μg/plate (the maximum recommended concentration according to current regulatory guidelines), in the absence and in the presence of a rat liver metabolic activation system (S-9).

Justification for classification or non-classification

Genetic toxicity - in vitro - It was concluded that the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains tested. These conditions included treatments at concentrations up to 5000 μg/plate (the maximum recommended concentration according to current regulatory guidelines), in the absence and in the presence of a rat liver metabolic activation system (S-9). The substance does not meet the criteria for classification in accordance with GHS or Regulation (EC) No 1272/2008 (CLP).