2-phenylimidazole

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Oct - 20 Nov 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted Jul 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-phenylimidazole

EC Number:

211-581-7

EC Name:

2-phenylimidazole

Cas Number:

670-96-2

Molecular formula:

C9H8N2

IUPAC Name:

2-phenyl-1H-imidazole

Test material form:

solid: flakes

Method

Target gene:

his operon

Species / strain

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:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and β-naphthoflavone.

Test concentrations with justification for top dose:

Preliminary toxicity study: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate with and without metabolic activation for tester strains TA98 and TA100

Based on the results of the preliminary toxicity study the following concentration levels were used for all tester strains in the main experiment:
Experiment I: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate with and without metabolic activation
Experiment II: 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine: -S9: 10 and 40 µg/plate for TA98 and TA1537, respectively; 2-aminoanthracene: +S9: 2.5 or 10 µg/plate for all strains

Remarks:

The biological activity of the metabolic activation system was investigated in a quality control using 2-aminoanthracene and benzo[a]pyrene.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation, Experiment I); preincubation (Experiment II)

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: Triplicates


DETERMINATION OF CYTOTOXICITY
- Method: Clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number if revertants occurs and/or
- a biologically relevant positive response for at least one of the dose group occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and TA102 the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.
According to the OECD guideline, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Results and discussion

Test results

Test results 1

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 with (Experiment II) and without metabolic activation (Experiment I and II)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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 with (Experiment II) and without metabolic activation (Experiment I and II)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate with and without metabolic activation in both Experiments (I and II).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

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 with (Experiment II) and without metabolic activation (Experiment I and II)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate without metabolic activation (Experiment I and II)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was observed in any tester strain used in Experiment I and II with and without metabolic activation, respectively.

RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity test was performed in tester strains TA98 and TA100. The following concentration levels were tested: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate with and without metabolic activation. The highest concentration level of 5000 µg/plate exhibited slight toxicity and was therefore chosen for the main experiment.

HISTORICAL CONTROL DATA
- Positive historical control data: The positive control values were within the range of the historical control data, therefore the test was considered to be valid.
- Negative (solvent/vehicle) historical control data: The negative control values were within the range of the historical control data, therefore the test was considered to be valid.

Any other information on results incl. tables

Table 2: Summary of results (Experiment I)

	TA98						TA100						TA1535						TA1537						TA102
	without metabolic activation			with metabolic activation			without metabolic activation			with metabolic activation			without metabolic activation			with metabolic activation			without metabolic activation			with metabolic activation			without metabolic activation			with metabolic activation
	Mean	SD	Mutation factor	Mean	SD	Mutation factor	Mean	SD	Mutation factor	Mean	SD	Mutation factor	Mean	SD	Mutation factor	Mean	SD	Mutation factor	Mean	SD	Mutation factor	Mean	SD	Mutation factor	Mean	SD	Mutation factor	Mean	SD	Mutation factor
Distilled water	22	5.7	0.9	27	7.8	1.1	112	7.5	1.5	73	11.8	1.0	16	2.5	1.0	13	2.9	0.8	29	0.6	1.0	20	2.1	1.0	293	0.6	1.1	313	36.1	1.1
DMSO	25	2.6	1.0	24	5.2	1.0	76	7.4	1.0	70	8.7	1.0	16	3.5	1.0	16	2.1	1.0	30	1.2	1.0	21	3.5	1.0	269	1.2	1.0	294	12.9	1.0
31.6 µg/plate	23	7.6	0.9	27	1.2	1.1	91	11.2	1.2	73	13.5	1.0	12	2.6	0.8	14	3.5	0.9	24	2.1	0.8	24	1.7	1.1	283	2.1	1.1	294	21.0	1.0
100 µg/plate	23	6.1	1.0	20	5.3	0.8	83	6.9	1.1	69	11.6	1.0	16	2.1	1.0	13	6.2	0.8	22	1.0	0.7	21	0.6	1.0	259	1.0	1.0	315	7.4	1.1
316 µg/plate	26	13.5	1.1	24	1.5	1.0	68	16.2	0.9	94	26.1	1.3	14	2.9	0.9	12	1.5	0.8	23	1.5	0.8	21	1.5	1.0	266	1.5	1.0	301	28.1	1.0
1000 µg/plate	26	5.0	0.7	25	6.0	1.0	68	4.0	0.9	88	22.9	1.3	14	3.5	0.9	10	0.0	0.6	20	1.5	0.7	20	4.0	0.9	244	1.5	0.9	276	2.1	0.9
2500 µg/plate	17	2.0	0.3	18	2.5	0.7	47	9.6	0.6	59	10.3	0.8	13	2.3	0.8	12	4.0	0.7	24	1.0	0.8	22	2.1	1.0	201	1.0	0.7	237	5.0	0.8
5000 µg/plate	6	3.5	0.5	12	6.1	0.5	15	3.5	0.2	47	23.8	0.7	13	2.5	0.8	5	4.7	0.3	15	7.0	0.5	22	2.6	1.0	138	7.0	0.5	192	22.3	0.7
4-NOPD	471	20.1	18.8	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	111	13.2	3.7	-	-	-	-	-	-	-	-	-
NA	-	-	-	-	-	-	736	157.8	9.7	-	-	-	1176	61.7	73.5	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
MMS	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	977	30.1	3.6	-	-	-
2-AA	-	-	-	2073	307.5	86.4	-	-	-	1522	53.2	21.7	-	-	-	249	32.0	15.9	-	-	-	108	14.0	5.1	-	-	-	806	8.5	1.7

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

NA: sodium azide

MMS: methylmethanesulphonate

2-AA: 2-Aminoanthracene

Table 2: Summary of results (Experiment II)

	TA98						TA100						TA1535						TA1537						TA102
	without metabolic activation			with metabolic activation			without metabolic activation			with metabolic activation			without metabolic activation			with metabolic activation			without metabolic activation			with metabolic activation			without metabolic activation			with metabolic activation
	Mean	SD	Mutation factor	Mean	SD	Mutation factor	Mean	SD	Mutation factor	Mean	SD	Mutation factor	Mean	SD	Mutation factor	Mean	SD	Mutation factor	Mean	SD	Mutation factor	Mean	SD	Mutation factor	Mean	SD	Mutation factor	Mean	SD	Mutation factor
Distilled water	26	5.0	1.0	26	5.3	1.1	98	4.2	1.1	87	7.5	1.1	22	0.6	1.0	15	3.2	0.9	17	1.0	1.0	20	1.5	1.0	275	37.8	1.0	370	53.1	1.2
DMSO	25	7.2	1.0	23	5.2	1.0	92	21.1	1.0	77	8.5	1.0	23	3.8	1.0	16	1.5	1.0	17	2.0	1.0	20	2.1	1.0	275	23.1	1.0	302	35.0	1.0
10.0 µg/plate	27	5.8	1.1	29	5.1	1.3	99	7.0	1.1	86	20.6	1.1	26	2.0	1.1	14	1.7	0.9	17	1.5	1.0	20	2.6	1.0	287	3.5	1.0	300	10.6	1.0
31.6 µg/plate	21	2.6	0.8	31	3.0	1.3	102	6.1	1.1	83	1.2	1.1	22	2.1	1.0	17	6.2	1.0	17	1.5	1.0	22	2.1	1.1	277	12.7	1.0	299	31.6	1.0
100 µg/plate	21	2.6	0.8	35	4.6	1.5	100	16.8	1.1	78	1.5	1.0	22	4.4	0.9	16	4.0	1.0	19	1.5	1.1	22	1.5	1.1	300	9.0	1.1	364	10.4	1.2
316 µg/plate	22	0.6	0.9	31	0.6	1.3	88	2.6	1.0	77	2.1	1.0	22	3.8	0.9	19	4.4	1.2	19	2.1	1.1	25	2.1	1.3	265	21.2	1.0	309	23.0	1.0
1000 µg/plate	22	7.5	0.9	28	6.0	1.2	84	9.3	0.9	85	6.8	1.1	27	3.1	1.1	18	5.2	1.1	18	1.5	1.1	21	3.2	1.1	277	36.2	1.0	357	29.0	1.2
2500 µg/plate	21	5.7	0.9	26	4.0	1.1	54	2.9	0.6	83	9.1	1.1	22	2.0	0.9	13	1.7	0.8	15	3.0	0.9	22	1.0	1.1	270	31.0	1.0	357	24.1	1.2
5000 µg/plate	11	4.9	0.4	7	4.0	0.3	18	16.3	0.2	26	7.9	0.3	6	1.2	0.2	4	1.5	0.2	4	1.5	0.2	8	1.5	0.4	133	17.2	0.5	182	82.2	0.6
4-NOPD	295	23.1	11.8	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	96	19.3	5.6	-	-	-	-	-	-	-	-	-
NA	-	-	-	-	-	-	634	44.7	6.9	-	-	-	836	107.0	35.8	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
MMS	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	692	41.2	2.5	-	-	-
2-AA	-	-	-	1693	477.0	73.6	-	-	-	1136	202.0	14.8	-	-	-	97	14.8	5.9	-	-	-	127	14.0	6.5	-	-	-	944	95.2	3.1

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

NA: sodium azide

MMS: methylmethanesulphonate

2-AA: 2-Aminoanthracene

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative