Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22.05.2019 - 14.10.2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 21, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 30, 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Hydrogen iodide
EC Number:
233-109-9
EC Name:
Hydrogen iodide
Cas Number:
10034-85-2
Molecular formula:
HI
IUPAC Name:
hydrogen iodide
Test material form:
liquid

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Due to migration, the value was transferred to one of the current document's attachments
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
To establish a dose response effect at least six dose levels with adequately spaced concentrations were tested. The maximum dose level was 5000 µg/plate.
Vehicle / solvent:
- Solvent used: deionised tap water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controls
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, 4-NOPD - without S9 mix: TA1537, TA 98 2-aminoanthracene, 2-AA - with S9 mix: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE:
- Test item preparation: On the day of the experiment, the test item was dissolved in deionised tap water. The test item was neutralised with NaOH2N. The pH value of the stock solution was 7 after neutralisation.

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 (plate incorporation test). 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. The pre-experiment is reported as main experiment I since the acceptance criteria were met.

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,
2000 μL Overlay agar

Experiment II (Pre-Incubation)
The following materials were mixed in a test tube and incubated at 37 °C for 60 minutes.
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.
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. 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.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Evident as a reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above 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.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate
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
Remarks:
at 5000 µg/plate without S9 mix
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 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate without S9 mix
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
Remarks:
at 2500 µg/plate without S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No precipitation of the test item occurred up to the highest investigated dose. 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 substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item 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.

Any other information on results incl. tables

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):

Strain

Experiment I

Experiment II

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

/

/

5000

5000

TA 1537

/

/

5000

/

TA 98

/

/

/

/

TA 100

/

/

5000

/

WP2 uvrA

/

/

2500 - 5000

/

/ = no toxic effects (induction factor ≥ 0.5)

 

Summary results from Experiment I:

Metabolic Activation

Test Group

Dose Level (per plate)

Revertant Colony Counts (Mean ± SD)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Without activation

Deionised water

16 ± 2

13 ± 7

27 ± 5

125 ± 6

56 ± 13

Untreated control

13 ± 5

12 ± 4

33 ± 8

130 ± 20

51 ± 16

Test Item

3 µg

14 ± 7

12 ± 4

33 ± 6

129 ± 5

57 ± 6

10 µg

15 ± 6

14 ± 4

35 ± 4

125 ± 13

62 ± 7

33 µg

12 ± 3

18 ± 1

29 ± 3

138 ± 11

59 ± 6

100 µg

11 ± 5

16 ± 3

35 ± 8

145 ± 7

60 ± 6

333 µg

13 ± 4

13 ± 1

32 ± 7

135 ± 12

45 ± 3

1000 µg

12 ± 5

16 ± 4

44 ± 7

140 ± 6

48 ± 8

2500 µg

9 ± 5

10 ± 2

32 ± 9

126 ± 13

40 ± 5

5000 µg

10 ± 5

13 ± 2

21 ± 5

105 ± 10

35 ± 11

NaN3

10 µg

1163 ± 29

1952 ± 95

4-NOPD

10 µg

295 ± 9

4-NOPD

50 µg

69 ± 3

MMS

2.0 µL

1050 ± 79

With activation

Deionised water

14 ± 5

13 ± 5

36 ± 1

125 ± 24

65 ± 11

Untreated control

15 ± 6

16 ± 5

38 ± 6

138 ± 7

57 ± 4

Test Item

3 µg

19 ± 2

16 ± 5

40 ± 11

131 ± 8

59 ± 7

10 µg

15 ± 4

12 ± 1

43 ± 2

153 ± 23

65 ± 11

33 µg

14 ± 4

12 ± 0

36 ± 9

147 ± 15

62 ± 6

100 µg

13 ± 6

10 ± 4

32 ± 5

129 ± 4

59 ± 8

333 µg

13 ± 4

11 ± 2

37 ± 4

129 ± 3

62 ± 3

1000 µg

10 ± 3

15 ± 5

40 ± 2

159 ± 21

63 ± 8

2500 µg

13 ± 3

12 ± 4

38 ± 7

143 ± 11

63 ± 10

5000 µg

10 ± 4

10 ± 4

40 ± 8

131 ± 9

62 ± 6

2-AA

2.5 µg

354 ± 55

405 ± 31

3308 ± 413

4604 ± 229

2-AA

10.0 µg

383 ± 30

Positive Controls: NaN3 – sodium azide, 2-AA – 2-aminoanthracene, 4-NOPD – 4-nitro-o-phenylene-diamine, MMS – methyl methane sulfonate.

 

Summary results from Experiment II:

Metabolic Activation

Test Group

Dose Level (per plate)

Revertant Colony Counts (Mean ± SD)

TA 1535

TA 1537

TA 98*

TA 100

WP2 uvrA

Without activation

Deionised water

14 ± 5

14 ± 4

26 ± 8

96 ± 8

44 ± 4

Untreated control

9 ± 5

16 ± 5

32 ± 1

102 ± 9

46 ± 11

Test Item

33 µg

13 ± 2

11 ± 5

32 ± 6

109 ± 8

38 ± 8

100 µg

15 ± 4

15 ± 4

28 ± 2

104 ± 1

46 ± 9

333 µg

10 ± 6

12 ± 3

28 ± 2

103 ± 19

50 ± 5

1000 µg

7 ± 3

9 ± 2

27 ± 9

104 ± 5

29 ± 4

2500 µg

10 ± 5

6 ± 1

28 ± 6

73 ± 11

12 ± 3

5000 µg

1 ± 1

3 ± 2

21 ± 2

2 ± 2

1 ± 1

NaN3

10 µg

1221 ± 59

594 ± 49

1770 ± 80

4-NOPD

10 µg

574 ± 24

4-NOPD

50 µg

76 ± 8

MMS

2.0 µL

646 ± 24

With activation

DMSO (Solvent control)

16 ± 2

13 ± 5

38 ± 3

100 ± 19

58 ± 6

Untreated control

18 ± 4

17 ± 4

26 ± 6

95 ± 5

51 ± 4

Test Item

33 µg

12 ± 3

12 ± 6

38 ± 12

101 ± 14

61 ± 6

100 µg

15 ± 5

16 ± 1

34 ± 7

98 ± 2

61 ± 6

333 µg

17 ± 6

12 ± 8

43 ± 3

100 ± 8

53 ± 6

1000 µg

15 ± 6

12 ± 2

28 ± 7

96 ± 14

49 ± 3

2500 µg

12 ± 3

10 ± 4

36 ± 12

89 ± 10

54 ± 6

5000 µg

6 ± 1

10 ± 3

25 ± 8

83 ± 20

52 ± 6

2-AA

2.5 µg

388 ± 55

295 ± 16

2449 ± 66

2825 ± 3

2-AA

10.0 µg

370 ± 20

Positive Controls: NaN3 – sodium azide, 2-AA – 2-aminoanthracene, 4-NOPD – 4-nitro-o-phenylene-diamine, MMS – methyl methane sulfonate.

*Due to irregular bacteria growth this part was repeated and is reported as part of experiment II

Applicant's summary and conclusion

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. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

A study was performed to investigate the potential of the test item 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 the Escherichia coli strain WP2 uvrA. The test was conducted according to OECD 471 (1997) and EU Method B.13/14 (30 May 2008). The assay was performed in three independent experiments. Experiment I and II were performed with and without liver microsomal activation. Each concentration, including the controls, was 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 in the overlay agar in the test tubes. 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. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in nearly all strains only in experiment II. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item 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.

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, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.