2-(2H-benzotriazol-2-yl)-4,6-bis(1-methyl-1-phenylethyl)phenol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline standard with acceptable restriction - S. typhimurium TA 102 or E. Coli to detect crosslinking/ oxidizing agents are not included in the panel of tested strains - no data on substance purity

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1982

Report date:

1982

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(lacking of test strains TA 102 or E. Coli, positive control in assays with metabolic activation tested in one strain only, confirmatory 2nd test performed only in TA 1537 and TA 1535)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(2H-benzotriazol-2-yl)-4,6-bis(1-methyl-1-phenylethyl)phenol

EC Number:

274-570-6

EC Name:

2-(2H-benzotriazol-2-yl)-4,6-bis(1-methyl-1-phenylethyl)phenol

Cas Number:

70321-86-7

Molecular formula:

C30H29N3O

IUPAC Name:

2-(2H-1,2,3-benzotriazol-2-yl)-4,6-bis(2-phenylpropan-2-yl)phenol

Details on test material:

- Batch No.: EN 24375

Specific details on test material used for the study:

- Batch No.: EN 24375

Method

Target gene:

Histidine operon

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of liver from rats induced with Aroclor 1254 with following cofactors at a ratio of 3:7

Test concentrations with justification for top dose:

25, 75, 225, 675 and 2,025 µg/plate

Vehicle / solvent:

- Vehicle: acetone
- Justification for choice of vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: cyclophosphamide (CP; 250 µg/plate in phosphate buffer, +S9, TA 1535 only); Daunorubicin-HCl (Drb-Hcl; 5 and 10 µg/plate in phosphate buffer, -S9, TA 98); N-methyl-N-nitro-N-nitrosoguanidine (MNNG; 3 and 5 µg/plate in phosphate buffer, - S9, TA 1535)

Remarks:

9(5)aminoacridine hydrochloride monohydrate (9-AA; 50 and 100 µg/plate in DMSO, -S9, TA 1537), 4 - nitroquinoline - N-oxide, NOQ, 0.125 and 0.25 µg/plate, -S9, TA 100)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation test)

DURATION
- Exposure duration: 48 hours at 37° C (in the dark)

NUMBER OF REPLICATIONS: 2 experiments; triplicate cultures/experiment

DETERMINATION OF CYTOTOXICITY
A preliminary toxicity test was carried out on strain TA 100 without microsomal activation with the concentrations ranging from 0.0001 to 2,000 ug/plate. The protocol used was the same as in the mutagenicity test, however, only two plates per concentration were used.

Evaluation criteria:

The test substance is generally considered to be nonmutagenic if the colony count in relation to the negative control is not doubled at any concentration.

Statistics:

When the colonies were counted, the arithmetic mean was computed.

Results and discussion

Test results

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the concentrations of 225 µg/plate and above the substance precipitated in soft agar

Any other information on results incl. tables

Table 1: Test results of experiment 1 (plate incorporation).

	[C] µg/plate	Revertants /plate (mean value of 3 plates)
		Base-pair substitution type				Frameshift type
		TA 1535		TA 100		TA 1537		TA 98
		-	+	-	+	-	+	-	+
NC	0	8	18	102	/	4	/	35	/
Drb-Hcl	5	/	/	/	/	/	/	314	/
	10	/	/	/	/	/	/	353	/
4-NOQ	0.125	/	/	298	/	/	/	/	/
	0.25	/	/	689	/	/	/	/	/
MNNG	3	1157	/	/	/	/	/	/	/
	5	1590	/	/	/	/	/	/	/
9AA	50	/	/	/	/	68	/	/	/
	100	/	/	/	/	976	/	/	/
CP	250	/	280	/	/	/	/	/	/
Test substance	0	17	11	5	93	29	8	104	49
	25	16	12	6	100	23	6	101	50
	75	13	19	8	103	30	5	117	50
	225	18	21	12	98	33	6	104	55
	675	21	22	9	96	32	6	107	50
	2,025	17	20	4	103	33	5	111	64

NC = Negative control

CP = Cyclophosphamide

Drb-Hcl = Daunorubicin-HC1

MNNG = N-methyl-N-nitro-N-nitrosoguanidine

9AA = 9-aminoacridine

NOQ = 4 - nitroquinoline - N-oxide

Table 2: Test results of experiment 2 (plate incorporation) 

	[C] µg/plate	Base-pair substitution type		Frameshift type
		TA 1535		TA 1537
		-	+	-	+
NC	0	7	10	6	/
MNNG	3	266	/	/	/
	5	1195	/	/	/
9AA	50	/	/	25	/
	100	/	/	389	/
CP	250	/	412	/	/
Test substance	0	11	13	8	5
	25	13	10	6	4
	75	12	12	5	6
	225	8	12	5	6
	675	14	11	3	6
	2,025	14	9	6	4

NC = Negative control

CP = Cyclophosphamide

MNNG = N-methyl-N-nitro-N-nitrosoguanidine

Applicant's summary and conclusion

Conclusions:

negative with metabolic activation
negative without metabolic activation