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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2004-12-08 to 2004-12-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Read-across from imidazole hydrobromide to imidazole hydroiodide is justified since both substances only differ in the respective counterion iodide or bromide. Both anions iodide and bromide are quite similar concerning toxicological behaviour and thus do not influence the toxicological results received.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Deviations:
yes
Remarks:
growth phase not mentioned; not clear if amino acid requirement was tested; number of cells per culture were not stated
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2002-01-14
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1H-Imidazole hydroiodide
Cas Number:
68007-08-9
Molecular formula:
C3H4N2*HI
IUPAC Name:
1H-Imidazole hydroiodide
Test material form:
solid
Details on test material:
- Name of test material (as cited in study report): Imidazolhydroiodid
- State of aggregation: colourless - slight yellow solid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, light protected

Method

Target gene:
TA1537: his C 3076
TA98: his D 3052
TA1535 & TA100: his G 46
WP2 uvrA: trp-
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:
S9-mix (containing 15 % S9): 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, and 5 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4
Test concentrations with justification for top dose:
Pre-experiment/Experiment 1: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate (with and without metabolic activation)
Experiment 2: 33, 100, 333, 1000, 2500, and 5000 µg/plate (with and without metabolic activation)
In the pre-experiment no toxic effects were observed and 5000 μg/plate were chosen as maximal concentration.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: solvent was chosen because of its solubility properties (Maron et al., 1981)*.

*Reference:
Maron, D.M., J. Katzenellenbogen and B.N. Ames, (1981) Compantibility of organic solvents with the Salmonella/Microsome Test. Mutation Res. 88, 343 - 350.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 2-aminoantracene (2-AA) & 4-nitro-o-phenylene-diamine (4-NOPD)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation; pre-experiment/experiment 1) and preincubation (experiment 2)

PRE-EXPERIMENT/EXPERIMENT 1:
To evaluate the toxicity of the test item a pre-experiment was performed with all strains. Eight concentrations were tested for toxicity and mutation induction with three plates each.

The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 μL test solution at each dose level, negative control or 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
After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.

Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment 1, if the following criteria are met: evaluable plates (>0 colonies) at five concentrations or more in all strains used.
The pre-experiment is reported as experiment 1 since no toxic effects were observed and 5000 μg/plate were chosen as maximal concentration.

EXPERIMENT 2
In the pre-incubation assay 100 μL test solution, 500 μL S9 mix/S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and shaken 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 selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.

NUMBER OF REPLICATIONS: triplicates/strain/dose level (incl. controls)

EVALUATION:
The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB 7BN, UK) with the software program Ames Study Manager.
Rationale for test conditions:
In the pre-experiment no toxic effects were observed and 5000 μg/plate were chosen as maximal concentration.
No precipitation of the test item occurred up to the highest investigated dose.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA98, TA100, and WP2 uvrA) or thrice (strains TA1535 and TA1537) 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.
Statistics:
No statistical evaluation of the data is required.

Results and discussion

Test results
Key result
Species / strain:
other: Salmonella typhimurium TA98, TA100, TA1535, and TA1537 as well as E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- precipitation: no precipitation of the test item occurred up to the highest investigated dose.

PRE-EXPERIMENT/EXPERIMENT 1 & EXPERMENT 2
- plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in both experiments.
- no toxic effects, evident as a reduction in the number of revertants, 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 the test item at any dose level, neither in the presence nor absence of metabolic activation. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
- positive controls showed a distinct increase of induced revertant colonies.
- number of colonies exceeded the laboratory's historical control range in the negative control of strain WP2 uvrA with and without metabolic activation in experiment 1, and in the solvent control of strain TA1535 without metabolic activation in experiment 2. In experiment 2, with metabolic activation, the number of colonies did not quite reach the lower limit of the laboratory's historical control data in strain TA100 (negative control). Since these deviations are rather small, these effects are judged to be based on biologically irrelevant fluctuations in the number of colonies and have no impact on the outcome of the study.
- historical range of positive controls was exceeded with metabolic activation in strains TA1535, TA1537, and TA98 in experiment 1 and in strain TA 100 in experiment 2. This effect indicates the sensitivity of the strains rather than compromising the assay.

Please also refer to the field "Attached background material" below.

HISTORICAL CONTROL DATA
- Positive historical control data / Negative (solvent/vehicle) historical control data: please refer to Table 1 in the field "Any other information on results incl. tables" below.

Any other information on results incl. tables

Table 1: Historical control data

Strain

 

without S9 mix

with S9 mix

 

Mean

SD

Min

Max

Mean

SD

Min

Max

TA1535

Solvent control

20

6

9

30

18

10

7

39

Negative contol

18

5

10

29

18

10

9

38

Positive control

3042

756

1003

3618

357

111

172

476

TA1537

Solvent control

11

7

4

29

18

9

6

31

Negative contol

12

6

5

29

18

6

8

29

Positive control

97

21

52

191

141

47

94

380

TA98

Solvent control

24

9

14

58

37

13

21

57

Negative contol

26

10

15

52

43

15

17

64

Positive control

379

98

137

976

1239

510

229

2566

TA100

Solvent control

121

29

91

198

149

36

109

281

Negative contol

141

23

101

189

147

43

103

254

Positive control

2089

408

1262

2872

921

346

546

2589

WP2 uvrA

Solvent control

50

16

34

65

53

17

34

65

Negative contol

51

10

38

63

50

13

35

60

Positive control

536

294

320

1976

346

38

221

415

 

 

Applicant's summary and conclusion

Conclusions:
The substance tested non-mutagenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.