1H-Imidazole, hydrobromide (1:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay/Ames test: negative (OECD 471; GLP)

Link to relevant study records

Reference

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.

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

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

Target gene:

TA1537: his C 3076
TA98: his D 3052
TA1535 & TA100: his G 46
WP2 uvrA: trp-

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.

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.

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.

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

 

 

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro

The substance was not observed to be mutagenic in a reliable bacterial reverse mutation assay (OECD 471).

Justification for classification or non-classification

Genetic toxicity in vitro

The substance should not be considered to have a mutagenic potential based on a bacterial reverse mutation assay (OECD 471). The substance does not require classification according to Regulation (EC) No 1272/2008 and subsequent adaptations.