α-phenyl-1H-benzimidazole-2-methanol

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:

08 March 2018 - 16 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ISO/IEC 17025:2005 (ISO/IEC, 2005)

Version / remarks:

2005

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-phenyl-1H-benzimidazole-2-methanol

EC Number:

200-073-0

EC Name:

α-phenyl-1H-benzimidazole-2-methanol

Cas Number:

50-97-5

Molecular formula:

C14H12N2O

IUPAC Name:

α-phenyl-1H-benzimidazole-2-methanol

Test material form:

solid

Details on test material:

Storage Conditions:Stable at ambient temperature (10-30°C). Store in darkness; may be used/formulated in light.

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

10.0, 33.3, 100, 333, 1000, 3333 and 5000 μg per plate.Doses selected for the mutagenicity assay are based on data generated in BioReliance study AF02PT.502.BTL, details was not documented in the study report

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: guideline recommended

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene: 1.0 μg/plate for TA98, TA1535, 2.0 μg/plate forTA100, TA1537 and 15 μg/plateWP2 uvrA in S9.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Duplicate
- Number of independent experiments: one – Plate incorporation method

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 50.0 μL of tester strain (cells seeded)
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: 50.0 μL of the test substance dilution were added to 2.0 mL of molten selective top agar at 45±2°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay had solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: n/a
- Exposure duration/duration of treatment: 48 to 72 hours at 37±2°C
- Harvest time after the end of treatment (sampling/recovery times): not stated

Rationale for test conditions:

The study was based on the in vitro (plate incorporation) technique described by Ames et al (1975), Maron and Ames (1983), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item. These strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium. Using these strains of Salmonella typhimurium revertants may be produced after exposure to a chemical mutagen, which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted. Additionally, a mutant strain of Escherichia coli (WP2uvrA) which requires tryptophan and can be reverse mutated by base substitution to tryptophan independence is used to complement the Salmonella strains.

Evaluation criteria:

For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance as specified below:

1. Strains TA1535 and TA1537 – considered positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.

2. Strains TA98, TA100 and WP2 uvrA - considered positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.

Statistics:

Not stated

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MTEST-SPECIFIC CONFOUNDING FACTORS: 
- Data on pH:Not specified
- Data on osmolality:Not specified
- Possibility of evaporation from medium:Not specified
- Water solubility:Not specified
- Precipitation and time of the determination: Precipitate was observed at 5000 μg per plate with all conditions.
- Definition of acceptable cells for analysis: All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
- Other confounding effects: no



RANGE-FINDING/SCREENING STUDIES (if applicable): Was not conducted



STUDY RESULTS
- Concurrent vehicle negative and positive control data: Both with historical control range


For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible: Yes in TA98, the dose dependent increases were also outside the historical control limit for this tester strain.
- Statistical analysis; not stated
- Any other criteria: not stated

Ames test:
- Signs of toxicity: Toxicity was observed beginning at 5000 μg per plate in TA1535 , 3333 in WP2 uvrA with and without S9 and 5000 μg per plate in TA1537 - S9
- Individual plate counts: yes
- Mean number of revertant colonies per plate and standard deviation: yes



Remarks on result:

other: TX16797induce reverse mutations at selected loci of Salmonella typhimurium (TA98) in the presence of metabolic activation

Any other information on results incl. tables

TABLE 1. Mutagenicity Assay without S9 activation

Strain	Substance	Dose level per plate	Mean Standard revertants	Deviation per plate	Ratio treated / solvent	Individual revertant colony counts and background codes
TA98	TX16797	5000 μg	11	3	0.5	15A1NP,9A1NP, 9A1NP
		3333 μg	12	4	0.6	14A, 7A, 14A
		1000 μg	23	5	1.1	17A, 24A, 27A
		333 μg	17	3	0.8	18A, 14A, 19A
		100 μg	20	5	1.0	24A, 21A, 15A
		33.3 μg	20	3	1.0	17A, 19A, 23A
		10.0 μg	16	2	0.8	14A, 16A, 18A
	DMSO	50.0 μL	21	4		22A, 17A, 24A
TA100	TX16797	5000 μg	53	12	0.6	52A2 NP, 42A2 NP, 66A2NP
		3333 μg	61	3	0.7	62A, 63A, 58A
		1000 μg	109	15	1.2	91A, 119A, 116A
		333 μg	105	12	1.2	111A, 112A, 91A
		100 μg	90	15	1.0	91A, 75A, 104A
		33.3 μg	96	23	1.1	123A, 83A, 82A
		10.0 μg	83	7	0.9	82A, 76A, 90A
	DMSO	50.0 μL	89	10		83A, 101A, 84A
TA1535	TX16797	5000 μg	3	3	0.3	6A1NP,3A1NP,1A1 NP
		3333 μg	7	5	0.6	7A, 11A, 2A
		1000 μg	16	3	1.5	19A, 15A, 14A
		333 μg	11	2	1.0	13A, 9A, 11A
		100 μg	14	1	1.3	15A, 13A, 14A
		33.3 μg	17	5	1.5	17A, 22A, 13A
		10.0 μg	12	4	1.1	9A, 17A, 10A
	DMSO	50.0 μL	11	4		11A, 8A, 15A
TA1537	TX16797	5000 μg	2	2	0.3	0A1NP,3A1NP,3A1 NP
		3333 μg	4	3	0.6	3A, 8A, 2A
		1000 μg	8	3	1.1	7A, 6A, 11A
		333 μg	7	2	1.0	8A, 7A, 5A
		100 μg	9	4	1.3	6A, 14A, 8A
		33.3 μg	7	2	1.0	7A, 5A, 9A
		10.0 μg	7	2	1.0	5A, 7A, 8A
	DMSO	50.0 μL	7	4		3A, 9A, 10A
WP2uvrA	TX16797	5000 μg	4	1	0.1	3A1NP,5A1NP,5A1 NP
		3333 μg	6	1	0.2	7A, 7A, 5A
		1000 μg	15	3	0.5	13A, 18A, 15A
		333 μg	23	4	0.8	26A, 19A, 24A
		100 μg	24	2	0.9	23A, 27A, 23A
		33.3 μg	27	4	1.0	32A, 25A, 24A
		10.0 μg	24	5	0.9	21A, 29A, 21A
	DMSO	50.0 μL	28	3		30A, 29A, 25A
TA98	2NF	1.00 μg	103	9	4.9	109A, 107A, 93A
TA100	SA	1.00 μg	656	45	7.4	678A, 686A, 604A
TA1535	SA	1.00 μg	546	8	49.6	547A, 538A, 553A
TA1537	9AA	75.0 μg	637	195	91.0	422A, 684A, 804A
WP2uvrA	MMS	1000 μg	455	35	16.3	415A, 477A, 474A
Key to Positive Controls				Key to Plate Postfix Codes		Key to Automatic Count Flags
2NF 2-nitrofluorene, SA sodium azide 9AAD 9-Aminoacridine MMS methyl methanesulfonate				NP = Non-interfering 1= particulate Normal background		A= Automatic count

TABLE 2. Mutagenicity Assay with S9 activation

Strain	Substance	Dose level per plate	Mean Standard revertants	Deviation per plate	Ratio treated / solvent	Individual revertant colony counts and background codes
TA98	TX16797	5000 μg	194	57	6.5	253A1 NP, 189A1 NP, 140A1 NP
		3333 μg	190	30	6.3	188A, 161A, 221A
		1000 μg	274	54	9.1	317A, 292A, 214A
		333 μg	95	18	3.2	79A, 92A, 115A
		100 μg	46	9	1.5	35A, 52A, 50A
		33.3 μg	32	4	1.1	36A, 29A, 31A
		10.0 μg	25	5	0.8	27A, 19A, 29A
	DMSO	50.0 μL	30	5		31A, 35A, 25A
TA100	TX16797	5000 μg	85	19	0.8	84A2 NP, 105A2 NP,67A2NP
		3333 μg	81	28	0.7	93A, 49A, 101A
		1000 μg	119	10	1.1	122A, 108A, 127A
		333 μg	101	18	0.9	88A, 93A, 122A
		100 μg	102	3	0.9	105A, 99A, 101A
		33.3 μg	102	6	0.9	106A, 105A, 95A
		10.0 μg	104	6	1.0	100A, 111A, 101A
	DMSO	50.0 μL	109	3		109A, 111A, 106A
TA1535	TX16797	5000 μg	2	1	0.2	3A1NP,2A1NP,2A1 NP
		3333 μg	5	4	0.4	9A, 2A, 5A
		1000 μg	9	1	0.8	10A, 9A, 9A
		333 μg	8	2	0.7	7A, 10A, 6A
		100 μg	12	5	1.0	9A, 9A, 18A
		33.3 μg	9	6	0.8	16A, 5A, 7A
		10.0 μg	12	3	1.0	14A, 13A, 9A
	DMSO	50.0 μL	12	2		14A, 10A, 13A
TA1537	TX16797	5000 μg	8	1	0.8	8A1NP,9A1NP,8A1 NP
		3333 μg	7	3	0.7	6A, 10A, 5A
		1000 μg	11	2	1.1	11A, 10A, 13A
		333 μg	9	3	0.9	7A, 13A, 8A
		100 μg	13	3	1.3	11A, 11A, 16A
		33.3 μg	10	5	1.0	6A, 8A, 15A
		10.0 μg	6	1	0.6	6A, 7A, 5A
	DMSO	50.0 μL	10	1		10A, 11A, 9A
WP2uvrA	TX16797	5000 μg	8	3	0.4	7A1NP,11A1NP, 6A1 NP
		3333 μg	8	4	0.4	13A, 7A, 5A
		1000 μg	18	3	0.8	17A, 16A, 21A
		333 μg	25	1	1.1	25A, 24A, 25A
		100 μg	26	4	1.2	31A, 23A, 25A
		33.3 μg	27	8	1.2	30A, 34A, 18A
		10.0 μg	23	2	1.0	21A, 23A, 24A
	DMSO	50.0 μL	22	5		27A, 17A, 22A
TA98	2AA	1.00 μg	242	28	8.1	274A, 223A, 230A
TA100	2AA	2.00 μg	799	40	7.3	845A, 776A, 776A
TA1535	2AA	1.00 μg	78	11	6.5	79A, 89A, 67A
TA1537	2AA	2.00 μg	44	11	4.4	41A, 35A, 57A
WP2uvrA	2AA	15.0 μg	350	18	15.9	332A, 352A, 367A
Key to Positive Controls				Key to Plate Postfix Codes		Key to Automatic Count Flags
2-aminoanthracene				NP = Non-interfering 1= particulate Normal background		A= Automatic count

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test item did cause a positive mutagenic response with tester strain TA98 in the presence of S9 activation.

Executive summary:

OECD 471 ( 2018): The test substance was tested to evaluate its mutagenic potential using the plate incorporation method by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

Doses selected for the mutagenicity assay were 10.0, 33.3, 100, 333, 1000, 3333 and 5000 μg per plate, based on data generated in BioReliance study AF02PT.502.BTL (detail was not included in the report). The maximum dose of 5000 μg per plate was achieved using a concentration of 100 mg/mL and a 50.0 μL plating aliquot.  All criteria for a valid study were met as described in the protocol.

The test item  in DMSO, at concentrations of 0.188 and 92.4 mg/mL, was stable at room temperature for at least 3.0 hours. No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.

Precipitate was observed at 5000 μg per plate with all conditions. Toxicity was observed beginning at 5000 μg per plate in TA1535, 3333  in WP2 uvrA with and without S9 and 5000 μg per plate  in TA1537 - S9. Positive mutagenic responses were observed (9.1-fold, maximum increase) with tester strain TA98 in the presence of S9 activation. The dose dependent increases were also outside the historical control limit for this tester strain. The study was concluded to be positive without conducting a confirmatory (independent repeat) assay because the results were clearly positive; hence, no further testing was warranted.