2-tert-butylphenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two Ames tests: Schöberl 1990 study and Noda 2001 study. Two in vitro chromosome abberration assays: Krueger 1998 study and Noda 2001 study.

Link to relevant study records

Reference

Reference 1

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.

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

Species / strain / cell type:

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

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

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

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.

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

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

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

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

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.

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

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).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Japanese Ministry of Health study. Well documented study performed to the principles of OECD guideline 471. No GLP status.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and 5,6-benzoflavone induced rat liver S9

Test concentrations with justification for top dose:

The test solutions were prepared at the time of use.
Dose-finding test - 50 to 5000 μg/plate.
Main test - 200 μg/plate at maximum and 100, 50, 25, 12.5 and 6.25 μg/plate using a common ratio of 2.

Vehicle / solvent:

DMSO
Test material not soluble in water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

furylfuramide

other: 2-aminoanthracene

Details on test system and experimental conditions:

The study was conducted by the pre-incubation method.

To a test tube, 0.1 mL of the solvent, test article solution or positive control article solution, 0.5 mL of 0.1 M sodium phosphate buffer (pH 7.4) for the direct method or 0.5 mL of S9 mix for the metabolic activation method, and then 0.1 mL of bacterial suspension were added and cultured with
shaking for 20 minutes at 37°C. Then, the mixture to which 2 mL of top agar was added and warmed to 45°C was overlaid on the plate. After the end of cultivation for 48 hours at 37°C, the number of revertant colonies was counted and the presence or absence of growth inhibition of the indicator bacterial strains was observed simultaneously using a stereomicroscope. The number of plates used was one for each dose concentration in the
dose selection test and three for each dose concentration in the main test. The main test was conducted twice using the same dose concentrations.

Evaluation criteria:

If the number of revertant colonies (mean value) in the test article-treated plate was at least twice that of the vehicle control plate and dose dependency and reproducibility of the results were observed, the test article was judged to be positive.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Growth inhibition of bacteria was observed at the dose levels of 200 μg/plate and above for all bacterial
strains irrespective of the presence or absence of metabolic activation, and slight growth inhibition was observed at 100 μg/plate in the direct
method for TA100, TA1535 and TA1537.

Conclusions:

Based on the results described above, it was judged that o-tert-butylphenol was negative for gene mutation inducibility under the conditions of this experiment

Executive summary:

In a study reported by the Japanese Ministry of Health, o-tert butyl phenol in DMSO (concentrations 6.25 to 200 μg/plate) was tested using the pre-incubation method in the absence (direct method) or presence (metabolic activation method) of S9 mix usingSalmonella typhimuriumTA100, TA1535, TA98, TA1537 andEscherichia coliWP2uvrAas index bacterial strains.

Under the conditions of this experiment, o-tert butyl phenol was not mutagenic.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998-09-08 to 1998-12-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media:
MEM4: MEM (Eagle) medium (lot #E05828-015 and E05828-116), purchased from PAA Laboratories GmbH, Germany, with 4 % fetal calf serum (FCS), 2 mM L-Glutamine, 100 IU/mL Penicillin, 100 µg/mL Streptomycin
MEM0: MEM (Eagle) medium with 2 mM L-Glutamine, 100 IU/mL Penicillin, 100 µg/mL Streptomycin (no FCS)
- Properly maintained: yes, stock culture maintained under liquid nitrogene
- Periodically checked for Mycoplasma contamination: not mentioned
- Periodically checked for karyotype stability: not mentioned
- Periodically "cleansed" against high spontaneous background: not applicable

Additional strain / cell type characteristics:

other: modal chromosome number of 22 and cell cycle length approx. 14 hours

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal fraction (S9 mix), Phenobarbital/ß-Naphthoflavone induced

Test concentrations with justification for top dose:

0 / 2.5 / 5 / 10 / 20 / 40 / 60 / 100 / 180 / 300 / 500 µg/mL in the first test with and without metabolic activation
0 / 0.5 / 1 / 2 / 4 / 6 / 10 / 20 / 40 / 60 µg/mL test substance without S9 mix
0 / 5 / 40 / 50 / 60 µg/mL in the second test with metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone, 1% (v/v)
- Justification for choice of solvent/vehicle: The test substance was soluble in acetone. In MEM medium containing 1% acetone, the solubility limit of the test subtance was determined to be 500 µg/mL (homogeneous emulsion, visual inspection).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

MEM0 medium containing 1% acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation Migrated to IUCLID6: 0.03, 0.04, 0.06 and 0.08 µg/mL, vehicle MEM4 medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

MEM0 medium containing 1% acetone plus S9 mix

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation Migrated to IUCLID6: 2 , 3 and 4 µg/mL, vehicle MEM0 medium

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
Cells were seeded in tissue culture dishes (1x10E5 cells per dish). After 24 h growth, the cells were treated with either 10 different test substance concentrations or the positive controls. MEM0 medium containing 1 % aceton served as the negative control. 3 h after test substance administration the medium was replaced by MEM4 medium. To arrest cells in metaphase, Colcemid (0.2 µg/mL final conc.) was added to the cultures 2 h prior to the cell preparation. Treatment was terminated 20 h after the start of exposure. For the termination of treatment cells were removed from the tissue culture dishes and transferred and centrifuged in tubes. 34 mM sodium citrate (prewarmed to 37 °C) was added. Hypotonic treatment was terminated after 8 min and 5 min centrifugation by fixation in methanol/glacial acetic acid (3 parts methanol, 1 part acetic acid, pre-cooled to 4 °C). 2 consecutive fixation steps each lasting 5 min by 800 rpm in a centrifuge were performed, afterwards cells were dropped onto slides, dried - and stained with giemsa.


DURATION
- Preincubation period: 24 hours
- Exposure duration: 3 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours


SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.2 µg/mL final conc.)
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: 2 per experimental point


NUMBER OF CELLS EVALUATED: 2000 per experimental point for determination of mitotic index; 200 metaphases per experimental point for analysis of chromosomal aberrations


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: Osmolality and pH of the highest test substance concentration


OTHER: none

Evaluation criteria:

The test is considered valid if the following criteria were met:
1. The number of cells with chromosomal aberrations (excl. gaps) in the negative control group falls within the normal range (< 5 %). The percentage of polyploid and endoreduplicated cells should be < 10 %.
2. Approx. 200 cells per treatment group (approx. 100 for each positive control) are analysable for chromosomal aberrations.
3. Treatment with positive controls (MMC and CP) in at least one concentration tested leads to clear increases in the frequency of cells with structurally altered chromosomes (>> 5 % excl. gaps).
The test chemical is to be considered clastogenic in this assay if
1. it induces chromosomal aberrations (excl. gaps) in a statistically significant manner in one or more concentrations
2. the induced proportion of aberrant cells at such test substance concentrations exceeds the normal range of the test system (i.e. >> 5 %)
3. positive results can be verified in an independent experiment.
The possible influence of pH, S9 mix or osmolality on the occurence of chromosomal aberrations will also be considered.

Statistics:

The proportion of cells that was treated with the test substance and harboured structural aberrations (excl. gaps) was compared with the corresponding proportion of the negative controls in the Chi-square test. Probability values of p < 0.05 were accepted as statistically significant

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: not applicable
- Water solubility: 2.3 g/L
- Precipitation: no precipitation up to 500 µg/mL
- Other confounding effects: none


RANGE-FINDING/SCREENING STUDIES: 10 different concentrations in the range of 2.5 to 500 µg/mL test substance were tested in the presence and absence of exogenous metabolic activation: 2.5 / 5 / 10 / 20 / 40 / 60 / 100 / 180 / 300 / 500 µg/mL. This concentrations were examined for mitotic indices. From these results the dose level reducing the metaphase index (i.e. the mitotic index) to approx. 50 % of the solvent control was used as the highest dose level for the metaphase analysis.
Concentrations chosen for scoring of chromosomal aberrations: control / 5 / 20 / 40 µg/mL test substance and Mitomycin C (0.03 µg/mL) as positive control in the experiment without S9 mix; control / 5 / 40 / 60 µg/mL test substance and Cyclophosphamid (3 µg/mL) as positive control with S9 mix in the first experiment.
Concentrations chosen for scoring of chromosomal aberrations in the repeat experiment: control / 4 / 20 / 40 µg/mL test substance and Mitomycin C (0.03 µg/mL) as positive control in the experiment without S9 mix; control / 5 / 40 / 50 / 60 µg/mL test substance and Cyclophosphamid (3 µg/mL) as positive control with S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: yes, the negative controls revealed chromosomal aberration frequencies which were consistent with spontaneous aberration frequencies for the V79 cells of the testing laboratory


ADDITIONAL INFORMATION ON CYTOTOXICITY: The experiments revealed a systematic influence of the test substance resulting in a reduction of the mitotic index (see tables below).
- With metabolic activation: 60 µg/mL (test #1), 40 µg/mL (test #2)
- Without metabolic activation: 40 µg/mL (test #1), 50 µg/mL (test #2)


GENOTOXIC EFFECTS:
- With metabolic activation: In the experiments with S9 mix the test compound showed a statistically and biologically significant induction of chromosomal aberrations in the proportion of cells excluding gaps. In test #1, the cells treated with the test compound revealed chromosomal aberration frequencies excluding gaps of 1.0 to 8.2 %. Therefore in test #2 an additional forth concentration was introduced. In the chromosomal aberrations study the positive result of test #1 could be repeated, chromosomal aberration frequencies excluding gaps of 3.5 to 9.5 % were reached. In both experiments the highest statistically significant increase in the proportion of cells with chromosomal aberrations (excl gaps) was not given in the highest but in the concentration underneath. However, these concentration showed a reduction of the mitotic index to approx. 50 % of the solvent control. The highest concentrations showed in both experiments in comparison with the second highest concentrations less reduction of the mitotic index. This is most likely the explanation for the highest statistically significant increase in these groups.
- Without metabolic activation: In both experiments (#1 and #2) treatment with o-tert-Butylphenol did not result in statistically or biologically significant increases in the frequency of cells with chromosomal aberrations excluding gaps.


FREQUENCY OF EFFECTS:
- with metabolic activation: 6.0 % chromosomal aberrations excl. gaps at the top concentration of 60 µg/mL and 8.2 % at 40 µg/mL were evaluated (1.0 % in the corresponding negative control) in test #1. In test #2 significant increases of the chromosomal aberation frequencies excluding gaps of 3.5 % (60 µg/mL), 9.5 % (50 µg/mL), 5.0 % (40 µg/mL) and 4.0 % (5 µg/mL), the corresponding negative control showed 0 %.

OTHER:
Due to the positive response of the test substance at the 20 hours sampling time, scoring of slides of the additional 28 hours sampling time was considered to be obsolete.

Table #1: Mitotic indices, range finding test #1

test substance	without S9 mix		with S9 mix
concentration [µg/mL]	Mitotic cells / cells scored	Mitotic index (% mean*)	Mitotic cell / cells sored	Mitotic index (% mean*)
Neg. control	146 / 2000	7.3	117 / 2000	5.9
2.5	- / -	not determined	- / -	not determined
5	170 / 2000	8.5	129 / 2000	6.5
10	- / -	not determined	- / -	not determined
20	143 / 2000	7.2	- / -	not determined
40	180 / 2000	9.0	55 / 2000	2.8
60	42 / 2000	2.1	77 / 2000	3.9
100	- / -	no cells	- / -	not enough cells
180	- / -	no cells	- / -	no cells
300	- / -	no cells	- / -	no cells
500	- / -	no cells	- / -	no cells
MMC 0.06	135 / 2000	6.8
CP 3			102 / 2000	5.1

Table #2: Mitotic indices test #2

test substance	without S9 mix		with S9 mix
concentration [µg/mL]	Mitotic cells /cells scored	Mitotic index (% mean*)	Mitotic cell /cells sored	Mitotic index (% mean*)
Neg. control	149 / 2000	7.5	85 / 2000	4.3
0.5	- / -	not determined
1	- / -	not determined
2	- / -	not determined
4	178 / 2000	8.9
5			113/ 2000	5.7
6	- / -	not determined
10	- / -	not determined
20	110 / 2000	5.5
40	74 / 2000	3.7	92 / 2000	4.6
50			41 / 2000	2.1
60	- / -	no cells	76 / 2000	3.8
MMC 0.03	77 / 2000	3.9
CP 3			43 / 2000	2.2

Table#3: Summary of data obtained in chromosomal aberration test #1

Treatment				Total # of mitotic cells scored	Proportion of cells with exchanges [%]	Proportion of cells with aberrations incl. gaps [%]	Proportion of cells with aberrations excl. gaps[%]	Significance$
µg/mL		Time [h]	S9 mix
0	Neg. control	20	-	200	0.5	8.0	0.5	-
5	test substance	20	-	200	0.0	8.0	0.5	ns
20	test substance	20	-	200	1.0	8.0	1.0	ns
40	test substance	20	-	200	0.5	6.0	1.0	ns
0.06	MMC	20	-	200	9.5	32.0	17.0	s
0	Neg. control	20	+	200	0.0	7.0	1.0	-
5	test substance	20	+	200	0.5	8.0	1.0	ns
40	test substance	20	+	200	2.9	18.3	8.2	***
60	test substance	20	+	200	2.0	12.5	6.0	*
3	CP	20	+	200	12.0	33.0	18.0	s

 $ ns = not significant s = significant, no statistical evaluation

* = statistically significant (0.01<P<0.05) *** = statistically significant (P<0.001)  

Table #4: Summary of data obtained in chromosomal aberration test #2

Treatment				Total # of mitotic cells scored	Proportion of cells with exchanges [%]	Proportion of cells with aberrations incl. gaps [%]	Proportion of cells with aberrations excl. gaps[%]	Significance $
µg/mL		Time [h]	S9 mix
0	Neg. control	20	-	200	0.0	6.5	0.0	-
4	test substance	20	-	200	0.5	5.0	1.0	ns
20	test substance	20	-	200	0.5	9.0	1.0	ns
40	test substance	20	-	200	0.0	12.0	2.0	ns
0.03	MMC	20	-	200	20.0	55.5	36.0	s
0	Neg. control	20	+	200	0.0	11.5	0.0	-
5	test substance	20	+	200	2.0	15.5	4.0	*
40	test substance	20	+	200	1.0	23.0	5.0	**
50	test substance	20	+	200	4.0	26.5	9.5	***
60	test substance	20	+	200	1.5	11.0	3.5	*
3	CP	20	+	200	18.0	56.5	33.5	s

$ ns = not significant s = significant, no statistical evaluation

* = statistically significant (0.01<P<0.05) ** = statisitically significant (0.001<P<0.01) *** = statisitcally significant (P<0.001)                                                                                                                                                                                                          

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
positive with metabolic activation

In the in vitro cytogenetic assay as described, o-tert.-Butylphenol did induce statistically and biologically significant increases in the chromosomal aberration frequency of V79 Chinese hamster cells. From the experiments performed, the author of the test report concluded that under the condition of this in vitro test system o-tert.-Butylphenol is a clastogenic agent. Nevertheless, o-tert.-Butylphenol is a highly cytotoxic substance, because of the relationship between cytotoxicity and clastogenic effect he cannot rule out that the clastogenic effect is secondary to the cytotoxicity.

Executive summary:

In an in vitro chromosome aberration assay, Chinese Hamster lung fibroblast cells (V79) were exposed to o-tert-butylphenol in DMSO at concentrations of

0 / 2.5 / 5 / 10 / 20 / 40 / 60 / 100 / 180 / 300 / 500 µg/mL in the first test with and without metabolic activation

0 / 0.5 / 1 / 2 / 4 / 6 / 10 / 20 / 40 / 60 µg/mL test substance without S9 mix

0 / 5 / 40 / 50 / 60 µg/mL in the second test with metabolic activation.

The study is well documented and follows the principles of OECD guideline method 473 with appropriate vehicle and positive controls.

O-tert butyl phenol did not induce any chromosome abberations in the absence of metabolic activation.

O-tert butyl phenol did induce chromosome abberrations and polyploidy in the presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

One in vivo bone marrow micronucleus assay: RTC study.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999-12-03 to 2000-01-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: HARLAN NOSSAN S.r.l., Carezzana (MI), Italy
- Age at study initiation: 5 - 6 weeks
- Weight at study initiation: 20.9 - 29.2 g
- Assigned to test groups randomly: animals were allocated to test groups, no further details mentioned
- Fasting period before study: overnight
- Housing: 5 animals/cage, by sexes, in clear polycarbonate cages measuring 35.5 x 23.5 x 19 cm with a stainless steel mesh lid and floor (Type 2b: Techniplast)
- Diet: Altromin MT diet ad libitum
- Water: drinking water ad libitum
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 10
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 1999-12-09 To: 1999-12-16

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: not stated
- Concentration of test material in vehicle: no data
- Amount of vehicle (if gavage or dermal): 10 ml/kg body weight
- Lot/batch no. (if required): batch no. 107H1649 obtained from Sigma Chemical Co.
- Purity: no data

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
no details mentioned

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

single treatment by oral gavage

Post exposure period:

none

Remarks:

Doses / Concentrations:
250, 500, 1000 mg/kg body weight
Basis:
no data

No. of animals per sex per dose:

preliminary toxicity test: 2
main assay: 5 for the low and mid dose group and the positive control (24 h sampling time); 10 for the vehicle control and the high dose group (24 and 48 h sampling times)

Control animals:

yes, concurrent vehicle

Positive control(s):

mitomycin C
- Justification for choice of positive control(s): not stated
- Route of administration: by gavage
- Doses / concentrations: solution of Mitomycin C (MMC, batch no. 088/AFA Kyowa Hakko Kogyo Co. Ltd., Tokyo) in sterile distilled water, concentration 3.0 mg/kg body weight

Tissues and cell types examined:

Preparation of the bone marrow were made on slides.
Poly- and normochromatic erythrocytes and micronucleated cells (poly- and normochromatic) were examined.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A dose-level equivalent to 80 % of the LD50 value was used as an estimate of the maximum tolerated dose-level.
- Criteria for selection of M.T.D.: Ratio of immature erythrocytes (PCE) to total erythrocytes (PCE+NCE), clinical signs and surviving of dosed animals. The M.T.D. should induce some tocix signs, such as the inhibition of bodyweight gain.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- Sampling times and number of samples: 24 h sampling time: five animals per sex from each group; 48 h sampling time: 5 animals per sex of the negative control and high dose group

DETAILS OF SLIDE PREPARATION:
- Bone marrow preparation: Animals of all groups were sacrified at appropriate sampling times. The femurs of animals were removed and bone marrow cells obtained by flushing with foetal calf serum. The cells were centrifuged and a concentrated suspension prepared to make smears on slides. These slides were air-dried and then stained with May-Gruenwald and Giemsa, and mounted with Eukitt. Three slides were made from each animal.


METHOD OF ANALYSIS:
- Slide evaluation: The slides were examined under low power (x 16 objective), one slide from each animal was selected according to staining and quality of smears. The slides were coded and where no depression of polychromatic erythrocytes was observed at least 2000 polychromatic cells per animal were examined for the presence of micronuclei at high power (x 100 objective, oil immersion). At the same time the numbers of normal and micronucleated normochromatic erythrocytes were also recorded.


OTHER:
Daily clinical observations of the treated animals were made for the justification for dose selection.

Evaluation criteria:

The test item is considered to induce micronuclei if a statistically significant increase in the micronuleus incindence in polychromatic erythrocytes (at P < 0.05) is observed in any treatment group, in the pooled data for both sexes, or for either sex considered separately.
Where increases in the incidence of micronuleated PCE`s were observed which were statistically significant, but fall within the range of negative control values within the testing laboratory, then concurrent and historical control data were used to denonstrate that these increases did not have biological significance. Historical controls were included in the test report.

Statistics:

Only counts obtained from polychromatic cells were subjected to statistical analysis. Using the original observations (and not the micronucleated frequencies per 2000 cells), a modified Chi-squared calculation was employed to compare treated and control groups. The degree of heterogeneity within each group was first calculated and where this was significant it was taken into account in the comparison between groups. Variance ratios or Chi-squared values are taken to show the significance of any difference between each treated group and the controls.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

at the highest dose level (1000 mg/kg bw)

Vehicle controls validity:

valid

Negative controls validity:

other: not performed

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range:
Toxicity test 1: 1000 and 2000 mg/kg body weight (male/female)
Toxicity test 2: 1300 (female only), 1600 (male/female) and 2000 (male only) mg/kg body weight
- Clinical signs of toxicity in test animals:
Toxicity test 1:
2000 mg/kg b.w. showed moribund animals, ataxia, hunched posture, reduced activity, pale appearance, cold to touch, swollen abdomen, increased activity, tremors, piloerection for both sexes.
1000 mg/kg b.w. showed ataxia, hunched posture, reduced activity, pale appearance, swollen abdomen, piloerection for both sexes.
female mice of the treatment with 2000 mg/kg bodyweight died, the surviving male mice were observed for 48 hours and sacrificed.
Toxicity test 2:
All animals of both sexes and all dose levels showed the following clinical signs: moribund animals, convulsions, hunched posture, reduced activity, pale appearance and piloerection.
Two male mice, treated with 2000 mg/kg b.w., one male mouse, treated with 1600 mg/kg b.w. and one female mouse, treated with 1300 mg/kg b.w. died. Animals were observed for 24 hours and sacrificed.
- Evidence of cytotoxicity in tissue analyzed: The ratio of immature erythrocytes (PCE) to total erythrocytes (PCE+NCE) showed mild depression bone marrow erythopoietic cell division following treatment with the test item, both in male and female animals.
- Rationale for exposure: Based on the results of the range finding study dose-levels of 250, 500 and 1000 mg/kg body weight were selected for the definitve study.



RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: No marked increases in the numbers of micronucleated PCE's were observed in any treatment group and any sampling time. Pronounced increases in the frequency of micronucleated PCE's were observed in the positive control group, indicating the correct functioning of the test system.
- Ratio of PCE/NCE: A moderate inhibitory effect to erythropoietic cell division was observed at the high dose-level in male animals, at 24 and 48 hours sampling time, and in female animals at 48 hour sampling time only.
- Appropriateness of dose levels and route: Clinical sighs, mortality and bone marrow cell toxicity provide evidence of bioavailibitity of the test substance and adequate exposure.
Clinical signs: 250 mg/kg b.w.: hunched posture and piloerection; 500 mg/kg b.w.: tremors, difficulty in movements and piloerection; 1000 mg/kg b.w.: hunched posture, difficulty in movements, tremors and piloerection. A number of male and female animals were found moribund the day of treatment, the surviving animals appeared to make full recovery before the final sampling time.
Mortality: Three female animals of the highest dose level (1000 mg/kg b.w.) died prior to the 24 hours sampling time, 2 of them were substituted by reserve animals.
- Statistical evaluation: No statistically significant increase in the incidence of micronucleated PCE¿s over the control value was observed at any sampling time for male and female animals combined. A statistically significant heterogeneity was observed within male animals from the high treatment group at 48 hour sampling time.

Table: Summary of incidences of micronucleated cells, both sexes

dose-level [mg/kg]	PCE/NCE ratio		incidence of micronucleated cells / 1000 cells
			polychromatic		normochromatic
	24 h	48 h	24 h	48 h	24 h	48 h
vehicle control	0.821	0.985	1.5 ± 0.1	1.4 ± 0.2	0.5 ± 0.2	0.6 ± 0.2
250	0.745		1.2 ± 0.2		0.7 ± 0.1
500	0.767		0.9 ± 0.2		0.5 ± 0.1
1000	0.636	0.563	1.1 ± 0.2	1.4 ± 0.4	0.6 ± 0.1	1.0 ± 0.2
positive control	0.749		10.4 ± 1.6		0.7 ± 0.2

Conclusions:

Interpretation of results (migrated information): negative
Following treatment with o-tert-Butylphenol, no statistically significant increase in the incidence of micronucleated PCE's over the control value was observed at any dose-level, at any sampling time.
Following treatment with the positive control Mitomycin-C, statistically significant increases in the incidence of micronucleated PCE's over the control values were observed, indicating the correct functioning of the test system.
It was concluded that, under the reported experimental conditions, o-tert-Butylphenol administered orally at the selected dose-levels to male and female mice, did not induce micronuclei in the polychromatic erythrocytes. Clinical signs and mortality observed at the highest dose-level, provide evidence of bioavailability of the test item and adequate exposure.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Two Ames tests for o-tert butyl phenol were both negative (not genotoxic) under the conditions of the studies.

Two in vitro chromosome aberration assays indicated o-tert butyl phenol did not induce chromosome aberrations in the absence of metabolic activation, but did induce chromosome aberrations and polyploidy in the presence of metabolic activation (rat liver S9 mix).

An in vivo bone marrow micronucleus assay was negative at doses of 500 mg/kg, 1000 mg/kg. There was good evidence from clinical observations that the material had entered the systemic circulation.

From the weight of evidence from these studies taken together, it is concluded that o-tert butyl phenol is not genotoxic/mutagenic.

Justification for selection of genetic toxicity endpoint
reliable in vivo study (Klimisch 1)

Justification for classification or non-classification

From the weight of evidence from two negative Ames tests, two in vitro chromosome aberrations assays (negative -S9, positive +S9) and an in vivo bone marrow micronucleus test (negative dosed at >500mg/kg with systemic exposure), the overall conclusion is that o-tert butyl phenol is not genotoxic/mutagenic.