2-tert-butylphenol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990-03-14 until 1991-04-23

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Limited number of values for assessment due to high cytotoxicity; poor documentation; strain to detect oxidising mutagens, cross-linking agents and hydrazines is missing; nevertheless considered sufficient for evaluation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-tert.-butylphenol

IUPAC Name:

2-tert.-butylphenol

Constituent 2

Reference substance name:

2-tert-butylphenol

EC Number:

201-807-2

EC Name:

2-tert-butylphenol

Cas Number:

88-18-6

Molecular formula:

C10H14O

IUPAC Name:

2-tert-butylphenol

Constituent 3

Reference substance name:

o.-tert.-butylphenol

IUPAC Name:

o.-tert.-butylphenol

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): 2-tert.-butylphenol
- Physical state: liquid
- Analytical purity: 99.97%
- Purity test date: 2014-05-07
- Lot/batch No.: 1419
- Expiration date of the lot/batch: 05/2015
- Stability under test conditions: stable
- Storage condition of test material: under N2 in tightly closed container at a cool, well ventilated place
- Colour: clear

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:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Arochlor 1254 induced liver S9 mix

Test concentrations with justification for top dose:

Plate incorporation test: 8 / 40 / 200 / 1000 / 5000 µg/plate
Pre-incubation test: 32 / 63 / 125 / 250 / 500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not mentioned

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Nitrofluorene (2.5 µg/plate) for the strains TA 98 and TA 1538; Sodium azide (2.5 µg/plate) for TA 100 and TA 1535; Aminoacredine (50 µg/plate) for TA 1537, Aminoanthracen (10 µg/plate) for TA 100

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Aminoanthracene (10 µg/plate) with strain TA 100

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation) and preincubation, performed in two independent tests


DURATION
- Preincubation period: 30 minutes
- Exposure duration: 96 hours


NUMBER OF REPLICATIONS: 3 per concentration


DETERMINATION OF CYTOTOXICITY
- Method: not mentioned


OTHER EXAMINATIONS:
- Other: Determination of the frequency of induced or spontaneous reversion to histidine independence with negative controls (H2O), solvent controls (DMSO), test substance concentrations and positive controls; determination of the titers of overnight cultures


OTHER: none

Evaluation criteria:

According to Ames a test article which causes no mutagenic effects at a concentration of 5000 µg/plate can be considered as non-mutagenic.

Statistics:

No statistics reported.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See Tables 1-4 below for concentrations with cytotoxicity.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See Tables 1-4 below for concentrations with cytotoxicity.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See Tables 1-4 below for concentrations with cytotoxicity.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See Tables 1-4 below for concentrations with cytotoxicity.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See Tables 1-4 below for concentrations with cytotoxicity.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not measured
- Effects of osmolality: not applicable
- Evaporation from medium: not applicable
- Water solubility: not mentioned
- Precipitation: no
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: not performed

COMPARISON WITH HISTORICAL CONTROL DATA: not performed

ADDITIONAL INFORMATION ON CYTOTOXICITY: See Tables 1-4 below for concentrations with cytotoxicity.

Any other information on results incl. tables

Table 1: Plate incorporation test: Number of revertants per plate (mean of 3 plates)

	[Strain TA 98]			[Strain TA 100]			[Strain TA 1535]
Conc. [unit]	- S9	+ S9	Cytotoxic (yes/no)	- S9	+ S9	Cytotoxic (yes/no)	- S9	+ S9	Cytotoxic (yes/no)
0*	22 ± 10	49 ± 4	no	266 ± 23	165 ± 17	no	4 ± 4	15 ± 5	no
8	23 ± 2	52 ± 6	no	276 ± 9	181 ± 8	no	2 ± 1	10 ± 4	no
40	15 ± 4	46 ± 7	no	224 ± 30	170 ± 23	no	0	20 ± 4	yes
200	8 ± 3	24 ± 1	no	237 ± 6	143 ± 5	no	0	14 ± 5	yes
1000	0	0	yes	0	0	yes	0	0	yes
5000	0	0	yes	0	0	yes	0	0	yes
Positive control	334 ± 79	not tested	no	302 ± 7	564 ± 167	no	250 ± 18	not tested	no

*solvent control with DMSO

Table 2: Plate incorporation test: Number of revertants per plate (mean of 3 plates)

	[Strain TA 1537]			[Strain TA 1538]
Conc. [unit]	- S9	+ S9	Cytotoxic (yes/no)	- S9	+ S9	Cytotoxic (yes/no)
0*	3 ± 3	18 ± 4	no	47 ± 31	52 ± 5	no
8	0	20 ± 7	yes	39 ± 11	54 ± 10	no
40	0	25 ± 10	yes	32 ± 2	48 ± 14	no
200	0	7 ± 4	yes	30 ± 9	54 ± 12	no
1000	0	0	yes	0	0	yes
5000	0	0	yes	0	0	yes
Positive control	408 ± 233	not tested	no	177 ± 15	not tested	no

*solvent control with DMSO

Table 3: Preincubation test: Number of revertants per plate (mean of 3 plates)

	[Strain TA 98]			[Strain TA 100]			[Strain TA 1535]
Conc. [unit]	- S9	+ S9	Cytotoxic (yes/no)	- S9	+ S9	Cytotoxic (yes/no)	- S9	+ S9	Cytotoxic (yes/no)
0*	34 ± 4	49 ± 11	no	120 ± 23	115 ± 16	no	15 ± 4	19 ± 2	no
32	40 ± 8	56 ± 2	no	114 ± 15	129 ± 3	no	18 ± 4	18 ± 4	no
63	33 ± 4	54 ± 4	no	130 ± 15	120 ± 31	no	13 ± 4	19 ± 6	no
125	0	54 ± 4	yes	0	120 ± 14	yes	0	14 ± 8	yes
250	0	51 ± 2	yes	0	93 ± 12	yes	0	0	yes
500	0	0	yes	0	0	yes	0	0	yes
Positive control	325 ± 24	not tested	no	578 ± 16	2531 ± 234	no	487 ± 39	not tested	no

*solvent control with DMSO

Table 4: Preincubation test: Number of revertants per plate (mean of 3 plates)

	[Strain TA 1537]			[Strain TA 1538]
Conc. [unit]	- S9	+ S9	Cytotoxic (yes/no)	- S9	+ S9	Cytotoxic (yes/no)
0*	12 ± 1	16 ± 2	no	43 ± 9	42 ± 4	no
32	12 ± 3	17 ± 6	no	35 ± 5	47 ± 9	no
63	9 ± 4	23 ± 5	no	36 ± 10	45 ± 5	no
125	0	19 ± 7	yes	31 ± 4	61 ± 9	no
250	0	3 ± 6	yes	0	50 ± 6	yes
500	0	0	yes	0	0	yes
Positive control	633 ± 106	not tested	no	155 ± 15	not tested	no

*solvent control with DMSO

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Increase of revertant colonies was observed neither at non-cytotoxic concentrations nor at the limit concentration. Therefore, based on the results from this Ames-test the test substance can be considered as non-mutagenic.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA 1535, TA 1537 and TA 1538 ofS. typhimurium were exposed to o-tert-butylphenol in DMSO at concentrations of 8, 40, 200, 1000, and 5000 µg/plate (plate incubation assay) and 32, 63, 125, 250, 500 µg/plate (pre-incubation assay) in the presence and absence of mammalian metabolic activation.

O-tert-butylphenol was tested up to cytotoxic concentrations and the limit concentration (5000 µg/plate). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is considered to be acceptable. This study satisfies the requirements for test guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data, except that a strain to detect oxidising mutagens, cross-linking agents and hydrazines is missing (e.g. TA102 or E. coli WP2 uvrA).