α-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol

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:

From August 6th to August 23rd, 2013.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

α-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol

EC Number:

246-042-5

EC Name:

α-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol

Cas Number:

24155-42-8

Molecular formula:

C11H10Cl2N2O

IUPAC Name:

1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethan-1-ol

Test material form:

solid: particulate/powder

Method

Target gene:

Histidine-requiring gene in four strains of S. typhimurium, and tryptophan requiring gene in E. coli WP2 uvrA.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Based on the results of the preliminary Range Finding Test (see 'Any other information on materials and methods including tables'), the following concentrations of the test item were prepared and used in the Initial and Confirmatory Mutation Tests:
- in Salmonella typhimurium strains: without metabolic activation: -S9 Mix: 500, 158, 50, 15.8, 5 and 1.6 μg/plate; with metabolic activation: +S9 Mix: 1581, 500, 158, 50, 15.8 and 5 μg/plate;
- in Escherichia coli WP2 uvrA: with and without metabolic activation: ±S9 Mix: 5000, 1581, 500, 158, 50 and 15.8 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The vehicle was compatible with the survival of the bacteria and the S9 activity and was chosen based on the results of the preliminary Solubility Test (the test item formed a clear solution at a concentration of 100 mg/mL in DMSO, se 'Any other information on materials and methods including tables').

Controls

Untreated negative controls:

yes

Remarks:

untreated control.

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylenediamine (4 µg/plate, source: Merck, Lot S6600094), 2-aminoanthracene (2 µg or 50 µg/plate for E.coli. Source: Sigma-Aldrich, Lot: STBB1901V)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
The study included a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method), and a Confirmatory Mutation Test (Pre-Incubation Method).
- Plate incorporation method: Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labeled. The test item and other components were prepared fresh and added to the overlay (45°C). This solution was mixed and poured on the surface of the properly labelled minimal agar plates (3 plates per control or concentration level). For activation studies, instead of phosphate buffer, 0.5 mL of the S9 Mix was added to each overlay tube. The entire test
consisted of non-activated and activated test conditions and each of them with the addition of negative and positive controls. The plates were incubated at 37°C for 48 hours.
- Pre-incubation method (main/confirmatory test): Before the overlaying of the test item, the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle). These tubes were gently mixed and incubated for 20 min at 37ºC in a shaking incubator. After the incubation period, 2 mL of molten top agar was added to the tubes, the content mixed and poured onto minimal glucose agar plates as described for the standard plate incorporation method. The entire test consisted of non-activation and activation test conditions, and each of them with the addition of negative and positive controls. After preparation, the plates were incubated at 37°C for about 48 hours.

DURATION
- Preincubation period: 20 min
- Exposure duration: 48h

NUMBER OF REPLICATIONS: 3.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth.

Rationale for test conditions:

The Preliminary Range Finding Test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix), at the concentrations of 5000, 1581, 500, 158, 50, 15.8 and 5 µg/plate of the test item. No insolubility of the test item was observed. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells were determined.
Strong inhibitory effect of the test item was obtained in both examined strains in the concentrations of 5000 and 1581 µg/plate, with and without metabolic activation (±S9 Mix). No bacterial growth was obtained in both strains at 5000 µg/plate (±S9 Mix). Strongly reduced revertant growth and additionally reduced background lawn development was noticed in both strains at 1581 µg/plate (±S9 Mix). Revertant colony numbers below the revertant colony numbers of the vehicle control and below the historical control data range; furthermore slightly reduced background lawn development indicated the inhibitory effect of the test item in S. typhimurium TA100 at the concentration of 500 μg/plate, without addition of metabolic activation (-S9 Mix). The obtained revertant colony numbers were lower than the revertant colony numbers of the vehicle control and were below the historical control data range indicating the cytotoxic effect of the test item in S. typhimurium TA98 at the concentration of 500 μg/plate, without addition of metabolic activation (-S9 Mix). In this experiment the observed revertant colony number increases were of minor intensity, far below the biologically relevant thresholds for being positive. Higher revertant colony counts within the corresponding historical control data ranges were obtained in S. typhimurium TA100 at 158, 50, 15.8 and 5 µg/plate (+S9 Mix). See 'Any other information on materials and methods incl. tables'.

Evaluation criteria:

- A test item is considered mutagenic if: a dose–related increase in the number of revertants occurs and/or a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
- An increase is considered biologically relevant if: in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control; in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control. According to the guidelines, 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.
- Criteria for a Negative Response: A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1581 and 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1581 and 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1581 and 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1581 and 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
- Effects of osmolality:
- Evaporation from medium:
- Water solubility:
- Precipitation:
- Definition of acceptable cells for analysis:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES: The Preliminary Range Finding Test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix), at the concentrations of 5000, 1581, 500, 158, 50, 15.8 and 5 µg/plate of the test item. No insolubility of the test item was observed. Strong inhibitory effect of the test item was obtained in both examined strains in the concentrations of 5000 and 1581 µg/plate, with and without metabolic activation (±S9 Mix). No bacterial growth was obtained in both strains at 5000 µg/plate (±S9 Mix). Strongly reduced revertant growth and additionally reduced background lawn development was noticed in both strains at 1581 µg/plate (±S9 Mix). Revertant colony numbers below the revertant colony numbers of the vehicle control and below the historical control data range; furthermore slightly reduced background lawn development indicated the inhibitory effect of the test item in S. typhimurium TA100 at the concentration of 500 μg/plate, without addition of metabolic activation (-S9 Mix). The obtained revertant colony numbers were lower than the revertant colony numbers of the vehicle control and were below the historical control data range indicating the cytotoxic effect of the test item in S. typhimurium TA98 at the concentration of 500 μg/plate, without addition of metabolic activation (-S9 Mix). In this experiment the observed revertant colony number increases were of minor intensity, far below the biologically relevant thresholds for being positive. Higher revertant colony counts within the corresponding historical control data ranges were obtained in S. typhimurium TA100 at 158, 50, 15.8 and 5 µg/plate (+S9 Mix).

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Historical control values for spontaneous revertants (revertants/plate) for positive reference control data without metabolic activation in the period of 2008 to 2012 were as follows: Salmonella typhimurium TA98: 116-735, TA100: 483-2047, TA1535: 376-1319, TA1537: 110-1499,Escherichia coli WP2 uvrA: 351-1247. The historical control values with metabolic activation were as follows: Salmonella typhimurium TA98: 430-2558, TA100: 497-2927, TA1535: 86-668, TA1537: 68-460, Escherichia coli WP2 uvrA: 147-533.
- Negative (solvent/vehicle) historical control data: Historical control values for spontaneous revertants (revertants/plate) for untreated control sample without metabolic activation were in the period of 2008 to 2012 were (as guide) as follows: Salmonella typhimurium TA98: 11-39, TA100: 68-173, TA1535: 3-23, TA1537: 2-19, Escherichia coli WP2 uvrA: 11-39. The historical control values with metabolic activation were as follows: Salmonella typhimurium TA98: 14-47, TA100: 77-174, TA1535: 4-23, TA1537: 2-20,
Escherichia coli WP2 uvrA: 19-53.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the Initial and Confirmatory Mutation Tests inhibitory effect of the test item was observed. The cytotoxicity was indicated by decreased revertant colony counts and affected background lawn development. In the performed experimental phases there were at least five analyzable concentrations and a minimum of three non-toxic dose levels at each tester strain.
- Measurement of cytotoxicity used: revertant colony counts and background lawn development.

Any other information on results incl. tables

Table 5. Summary Table of the Results of the Initial Mutation test.

Initial Mutation Test (Plate Incorporation Test)
Concentration µg/ plate	Salmonella typhimuriumtester strains																Escherichia coli WP 2uvrA
	TA 98				TA 100				TA 1535				TA 1537
	- S9		+ S9		- S9		+ S9		- S9		+ S9		- S9		+ S9		- S9		+ S9
Mean values of revertants per plate and Mutation Rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated control	20.3	1.17	29.7	1.02	74.7	0.99	118.0	1.08	5.3	1.23	8.0	0.89	4.7	0.74	8.7	1.24	22.3	0.93	34.0	0.96
DMSO control	17.3	1.00	29.0	1.00	75.3	1.00	109.7	1.00	4.3	1.00	9.0	1.00	6.3	1.00	7.0	1.00	24.0	1.00	35.3	1.00
Ultrapure Water control	–	–	–	–	75.7	1.00	–	–	5.3	1.00	–	–	–	–	–	–	24.3	1.00	–	–
5000	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	0.7	0.03	0.3	0.01
1581	–	–	0.3	0.01	–	–	2.7	0.02	–	–	0.3	0.04	–	–	0.3	0.05	13.0	0.54	17.7	0.50
500	8.0	0.46	23.3	0.80	58.3	0.77	92.3	0.84	6.3	1.46	11.3	1.26	0.7	0.11	4.3	0.62	15.7	0.65	33.7	0.95
158	22.7	1.31	28.7	0.99	76.3	1.01	112.3	1.02	5.3	1.23	8.3	0.93	6.7	1.05	6.7	0.95	22.7	0.94	33.0	0.93
50	23.7	1.37	24.3	0.84	71.0	0.94	111.7	1.02	8.7	2.00	7.0	0.78	6.7	1.05	8.7	1.24	26.0	1.08	35.3	1.00
15.8	13.0	0.75	27.0	0.93	68.7	0.91	108.0	0.98	3.7	0.85	7.0	0.78	7.3	1.16	7.3	1.05	17.3	0.72	29.7	0.84
5	18.7	1.08	28.3	0.98	60.7	0.81	105.7	0.96	4.7	1.08	8.0	0.89	6.0	0.95	5.7	0.81	–	–	–	–
1.6	19.0	1.10	–	–	66.3	0.88	–	–	5.3	1.23	–	–	6.0	0.95	–	–	–	–	–	–
NPD (4µg)	173.7	10.02	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2µg)	–	–	–	–	681.3	9.00	–	–	621.3	116.50	–	–	–	–	–	–	–	–	–	–
9AA (50µg)	–	–	–	–	–	–	–	–	–	–	–	–	259.7	41.00	–	–	–	–	–	–
MMS (2µL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	1137.3	46.74	–	–
2AA (2µg)	–	–	1493.3	51.49	–	–	1478.7	13.48	–	–	151.0	16.78	–	–	87.3	12.48	–	–	–	–
2AA (50µg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	373.0	10.56

 

Table 6. Summary Table of the Results of the Confirmatory Mutation test.

Confirmatory Mutation Test (Pre-Incubation Test)
Mean values of revertants per plate and Mutation Rate (MR)	Salmonella typhimuriumtester strains																Escherichia coli WP 2uvrA
	TA 98				TA 100				TA 1535				TA 1537
	- S9		+ S9		- S9		+ S9		- S9		+ S9		- S9		+ S9		- S9		+ S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	15.3	1.07	15.0	0.80	77.3	1.10	112.3	1.05	10.0	0.91	13.3	0.63	9.3	1.08	8.0	0.80	27.0	1.50	26.7	0.80
DMSO Control	14.3	1.00	18.7	1.00	70.3	1.00	107.3	1.00	11.0	1.00	21.0	1.00	8.7	1.00	10.0	1.00	18.0	1.00	33.3	1.00
Ultrapure Water Control	–	–	–	–	67.0	1.00	–	–	9.7	1.00	–	–	–	–	–	–	23.3	1.00	–	–
5000	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	0.0	0.00	0.0	0.00
1581	–	–	11.3	0.61	–	–	8.3	0.08	–	–	0.0	0.00	–	–	3.7	0.37	5.7	0.31	5.7	0.17
500	5.3	0.37	18.0	0.96	17.3	0.25	106.3	0.99	1.7	0.15	16.0	0.76	6.7	0.77	10.0	1.00	11.7	0.65	26.3	0.79
158	14.3	1.00	26.7	1.43	58.0	0.82	117.0	1.09	8.0	0.73	17.0	0.81	8.3	0.96	8.3	0.83	18.7	1.04	32.3	0.97
50	24.7	1.72	24.0	1.29	77.3	1.10	99.7	0.93	8.7	0.79	14.0	0.67	9.7	1.12	10.7	1.07	24.0	1.33	34.3	1.03
15.8	27.3	1.91	26.3	1.41	69.3	0.99	108.7	1.01	11.3	1.03	13.7	0.65	9.3	1.08	10.3	1.03	20.0	1.11	33.7	1.01
5	28.0	1.95	21.0	1.13	82.7	1.18	107.7	1.00	10.3	0.94	14.0	0.67	9.0	1.04	10.3	1.03	–	–	–	–
1.6	26.0	1.81	–	–	84.7	1.20	–	–	10.7	0.97	–	–	8.7	1.00	–	–	–	–	–	–
NPD (4µg)	230.7	16.09	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2µg)	–	–	–	–	494.0	7.37	–	–	407.0	42.10	–	–	–	–	–	–	–	–	–	–
9AA (50µg)	–	–	–	–	–	–	–	–	–	–	–	–	339.7	39.19	–	–	–	–	–	–
MMS (2µL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	574.0	24.60	–	–
2AA (2µg)	–	–	1160.7	62.18	–	–	1268.0	11.81	–	–	123.0	5.86	–	–	80.0	8.00	–	–	–	–
2AA (50µg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	160.0	4.80

Applicant's summary and conclusion

Conclusions:

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Therefore, the test item is not mutagenic.

Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay, according to OECD 471, under GLP conditions. The study included a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method), and a Confirmatory Mutation Test (Pre-Incubation Method). Based on the results of the Preliminary Range Finding Test, four histidine requiring strains of S. typhimurium (TA1535, TA 1537, TA 98, TA 100) and tryptophan requiring E.coli WP uvrA were tested in triplicate at the following concentrations: in Salmonella typhimurium strains: without metabolic activation: -S9 Mix: 500, 158, 50, 15.8, 5 and 1.6 μg/plate; with metabolic activation: +S9 Mix: 1581, 500, 158, 50, 15.8 and 5 μg/plate; in Escherichia coli WP2 uvrA: with and without metabolic activation: ±S9 Mix: 5000, 1581, 500, 158, 50 and 15.8 μg/plate. At the preparation of the test item stock solution a correction factor (based on active ingredient content) of 1.086 was taken into consideration. Vehicle (DMSO), untreated and positive controls were run in parallel. The revertant colony numbers of vehicle control (Dimethyl sulfoxide, DMSO) plates with and without S9 Mix were within the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Therefore, the test item is not mutagenic.