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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames: negative

CA: negative

HGPRT: negative

Link to relevant study records

Referenceopen allclose all

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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Batch-no.: 1437
Purity: min. 99%
Species / strain / cell type:
S. typhimurium, other: TA97a
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
S. typhimurium TA 1535
Metabolic activation:
with and without
Metabolic activation system:
S9 enzymes from the livers male Sprague-Dawley rats, treated with 500 mg Aroclor 1254/kg bw
Test concentrations with justification for top dose:
Toxicity test: 5000 / 1500 / 500 / 150 / 50 µg/plate
First experiment: 1500 / 500 / 150 / 50 / 15 µg/plate
second experiment: 1500 / 750 / 375 / 188 / 94 / 47 / 23 / 12 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: In a preliminary test, the solubility of the test item was determined in demineralised water and DMSO. The test item was only soluble in a concentration of 50 g/L in DMSO.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-1,2-phenylene, 20 µg/plate with strains TA97a, TA98 and TA102; Sodium Azide, 1 µg/plate with strains TA100 and TA1535
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Amino-anthracene, 1 µg/plate with strains TA97a, TA100, TA102 and TA1535; Benzo-a-pyrene, 20 µg/plyte with strain TA98
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) in the first experiment; preincubation in the second experiment

DURATION
- Preincubation period: 20 min (only in the second experiment)
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY:
- Method: In the pre-experiment: determination of titre (the test item was considered non-toxic, if the quotient titre/toxicity is below 2), in the main experiments: evaluation of background lawn, reduction in number of revertants in comparison to negativ/solvents control

OTHER EXAMINATIONS:
- Visual counting of mutant colonies, a spreadsheet software (Microsoft Excel) was used to calculate mean values and standard deviations.
- Quality control of bacterial strains: genotype confirmation for each batch of bacteria before stock culture preparation: all bacterial strains were tested for histidine requirement, ampicillin resistence, crystal violet sensitivity, UV sensitivity and spontaneous revertants, furthermore the following examinations were performed: determination of titre, toxicity control, sterility control and positive control

Evaluation criteria:
The colonies were counted visually, the numbers were recorded. A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control. The increase factor f(I) of revertant induction (mean revertants divided by mean spontaneous revertants) and the absolute number of revertants (revertants less mean spontaneous revertants) were also calculated.

A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate in at least one strain exceeding an increase factor of 2 (in tester strains TA 97a, TA98, TA100 and TA102) and an increase factor of 3 (in tester strain TA1535) as compared to the reversion rate of the solvent control can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.
Statistics:
not performed
Species / strain:
S. typhimurium, other: TA 97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in the preincubation experiment at 1500 and 750 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in the preincubation experiment at 1500 and 750 µg/plate
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:
in the preincubation experiment at 1500 and 750 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in the preincubation experiment at 1500 and 750 µg/plate
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:
in the preincubation experiment at 1500 and 750 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: not soluble in water
- Precipitation: Precipitated/undissolved test item was not observed at any of the concentrations tested.
- Other confounding effects: nothing mentioned

RANGE-FINDING/SCREENING STUDIES: A pre-experiment for toxicity was performed according to the plate incorporation method. The toxicity of the following concentrations were tested: 5000 / 1500 / 500 / 150 / 50 µg/plate. Toxicity was observed in all tester strains only in the highest concentration.

COMPARISON WITH HISTORICAL CONTROL DATA: Nearly all determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory, differences were only marginal and no critical impact on the outcome of the study was expected. All positive control showed mutagenic effects with and without metabolic activation.

Table #1: First Mutation Assay (Direct Plate Incorporation Method)

  TA 97a           TA 98               TA 100      
      - S9 mix + S9 mix     - S9 mix   + S9 mix     - S9 mix     + S9 mix    
Dose level[µg/plate]   Mean revertants per plate ± SD  f (I)  Mean revertants per plate ± SD  f (I)  Mean revertants per plate ± SD  f (I)  Mean revertantsper plate± SD  f (I)  Mean revertants per plate ± SD  f (I)  Mean revertants per plate ± SD  f (I)
H2O  120 ± 2.3   -  1115 ± 4.5  -  19 ± 6.1  -  17 ± 4.0  -  91 ± 1.0  -  123 ± 6.4  -
 DMSO  118 ± 4.2  -  116 ± 3.6  -  20 ± 2.6  -  22 ± 2.1  -  97 ± 9.5  -  114 ± 11.9  -
 1500  133 ± 22.0  1.13  115 ± 4.4  0.99  12 ± 2.0  0.60  13 ± 2.3  0.59  113 ± 5.2  1.16  115 ± 4.0  1.01
 500  116 ± 4.2  0.98  115 ± 4.7  0.99  13 ± 0.6  0.65  14 ± 1.7  0.64  117 ± 1.7  1.21  120 ± 5.5  1.05
 150  112 ± 1.7  0.95  129 ± 6.1  1.11  10 ± 3.5  0.50  15 ± 3.8  0.68  106 ± 14.6  1.09  100 ± 11.1  0.88
 50  123 ± 15.4  1.04  113 ± 7.6  0.97  13 ± 1.2  0.65  18 ± 0.6  0.82  129 ± 9.5  1.33  115 ± 18.2  1.01
 15  118 ± 4.6  1.00  115 ± 5.6  0.99  17 ± 2.5  0.85  20 ± 4.2  0.91  105 ± 11.4  1.08  94 ± 8.4  0.81
 Positive controls  497 ± 33.3  4.21  485 ± 22.0  4.18  493 ± 44.1  24.7  102 ± 10.3  4.64  731 ± 41.6  8.03  661 ± 31.1  5.80

f (I) = increase factor of revertant induction (mean revertants divided by mean spontaneous revertants)

Table #1 (continued): First Mutation Assay (Direct Plate Incorporation Method)

  TA 102           TA 1535          
  - S9 mix     + S9 mix     - S9 mix      + S9 mix   
Dose level [µg/plate]   Mean revertants per plate ± SD  f (I)  Mean revertants per plate ± SD  f (I)  Mean revertants per plate ± SD  f (I)  Mean revertants per plate ± SD  f (I)
 H2O  202 ± 8.7  -  199 ± 20.8  - 21 ± 1.0  -  22 ± 3.1  -
 DMSO  198 ± 15.6  -  237 ± 5.0  -  20 ± 2.6  -  17 ± 4.0  -
 1500  169 ± 12.2  0.85  229 ± 19.7  0.97  13 ± 3.8  0.65  13 ± 5.5  0.73
 500  197 ± 10.1  0.99  219 ± 23.2  0.92  14 ± 4.4  0.70  11 ± 2.1  0.65
 150  204 ± 17.4  1.03  219 ± 11.0  0.92  10 ± 0.6  0.50  13 ± 2.3  0.76
 50  188 ± 33.6  0.95  224 ± 28.3  0.95  15 ± 3.8  0.75  12 ± 2.5  0.71
 15  248 ± 45.4  1.25  208 ± 43.3  0.88  12± 2.5  0.60  14 ± 2.1  0.82
 Positiv controls  779 ± 209.8  3.93  955 ± 90.0  4.03  133 ± 16.3  6.33  112 ± 14.0  6.59

f (I) = increase factor of revertant induction (mean revertants divided by mean spontaneous revertants)

Table #2: Second Mutation Assay (Pre-incubation Method)

  TA 97a           TA 98               TA 100      
      - S9 mix + S9 mix     - S9 mix   + S9 mix     - S9 mix     + S9 mix    
Dose level [µg/plate]   Mean revertants per plate ± SD  f (I)  Mean revertants per plate ± SD  f (I)  Mean revertants per plate ± SD  f (I)  Mean revertants per plate ± SD  f (I)  Mean revertants per plate ± SD  f (I)  Mean revertants per plate ± SD  f (I)
H2O   129 ± 8.5  -  124 ± 16.9  -  14 ± 1.5  -  13 ± 0.0  -  135 ± 6.1  -  133 ± 13.1  -
 DMSO  109 ± 7.8  -  109 ± 6.5  -  12 ± 2.6  -  10 ± 0.0  -  132 ± 5.9  -  139 ± 8.1  -
 1500  0 ± 0.0  0  0 ± 0.0  0  0 ± 0.0  0  0 ± 0.0  0  0 ± 0.0  0  0 ± 0.0  0
 750  10 ± 1.0  0.09  12 ± 6.7  0.11  1 ± 1.7  0.08  7 ± 4.9  0.70  12 ± 4.2  0.09  9 ± 5.2  0.06
 375  129 ± 22.4  1.18  134 ± 13.3  1.23  10 ± 2.5  0.83  10 ± 0.6  1.00  40 ± 2.6  0.30  31 ± 14.6  0.22
 188  117 ± 10.7  1.07  135 ± 8.7  1.24  13 ± 1.0  1.08  12 ± 1.2  1.20  110 ± 5.1  0.83  108 ± 11.1  0.78
 94  107 ± 6.1  0.98  110 ± 1.0  1.01  11 ± 2.0  0.92  12 ± 2.1  1.20  122± 16.3  0.92  111 ± 5.5  0.80
 47  105 ± 5.5  0.96  104 ± 5.1  0.95  13 ± 3.5  1.08  11 ± 1.7  1.10  117 ± 1.2  0.89  115 ± 10.1  0.83
 23  112 ± 3.1  1.03    134 ± 12.5  1.23  15 ± 1.2  1.25 15  ± 3.1  1.50  110 ± 12.6  0.83  119 ± 23.6  0.86
12   109 ± 2.1  1.00  138 ± 12.9  1.27  10 ± 0.6  0.83  11 ± 1.2  1.10  130 ± 13.6  0.98  96 ± 14.2  0.69
 Positive controls  499 ± 44.1  4.58  456 ± 73.0  4.18  109 ± 1.2  9.08  99 ± 10.1  9.90  555 ± 36.1  4.11  717 ± 184.0  5.16

f (I) = increase factor of revertant induction (mean revertants divided by mean spontaneous revertants)

Table #3 (continued): Second Mutation Assay (Pre-incubation Method)

  TA 102           TA 1535          
  - S9 mix     + S9 mix     - S9 mix      + S9 mix   
Dose level [µg/plate]   Mean revertants per plate ± SD  f (I)  Mean revertants per plate ± SD  f (I)  Mean revertants per plate ± SD  f (I)  Mean revertants per plate ± SD  f (I)
 H2O  365 ± 17.0  -  370 ± 23.6  -  14 ± 4.0 - 10 ± 0.6  -
 DMSO  351 ± 68.9  -  397 ± 19.7  -  14 ± 3.1  -  14 ± 3.2  -
 1500  114 ± 17.5  0.32  7 ± 5.5  0.02  0 ± 0.0  0  3 ± 1.5  0.21
 750  107 ± 14.6  0.30  86 ± 11.0  0.22  4 ± 2.3  0.29  6 ± 0.0  0.43
 375  210 ± 20.0  0.60  210 ± 47.6  0.53  8 ± 1.5  0.57  10 ± 0.6  0.71
 188  267 ± 56.6  0.76  361± 27.2  0.91  15 ± 3.1  1.07  16 ± 2.6  1.14
 94  313 ± 31.9  0.89  363 ± 9.2  0.91  12 ± 3.1  0.86  12 ± 2.1  0.86
 47  361 ± 64.0  1.03  265 ± 28.4  0.67  16 ± 0.6  1.14  15 ± 3.1  1.07

23 

 275 ± 41.1

 0.78

 318 ± 56.0

 0.80

 11 ± 3.2

 0.79

 12 ± 2.6

 0.86

 12  421 ± 51.6  1.20  372 ± 65.5  0.94  17 ± 8.7  1.21  12 ± 4.0  0.86

 Positive

controls

 1076 ± 133.1

 3.07

 1071 ± 63.5

 2.70

 165 ± 39.3

 11.8

 134 ± 6.7

 9.57

f (I) = increase factor of revertant induction (mean revertants divided by mean spontaneous revertants)

Conclusions:
Interpretation of results (migrated information):
negative

The test item did not show mutagenic effects towards Salmonella typhimurium, strains TA97a, TA98, TA100, TA102 and TA1535. Therefore, no concentration-effect relationship could be determined. The test item 2,4-di-tert-butylphenol is considered as "not mutagenic under the conditions of the test":
Executive summary:

Two valid experiments were performed following OECD 471 and EU B.13/14 under the conditions of GLP.

First Experiment: On the base of a pre-test for toxicity, five concentrations of the test item, dissolved in DMSO (ranging from 15 to 1500 μg/plate) were used. Five genetically changed strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 (genetically manipulated) and TA1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system (S9-mix, rat liver S9-mix induced by Aroclor 1254) for 48 hours, using the plate incorporation method. None of the concentrations caused a significant increase in the number of revertant colonies in the tested strains. The test item did not show any mutagenic effects in the first experiment. The test item showed no precipitates on the plates in all tested concentrations. No signs of toxicity towards the bacteria could be observed. The sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

Second Experiment: To verify the results of the first experiment, a second experiment was performed, using eight concentrations of the test item (ranging from 12 to 1500 μg/plate) and a modification in study performance (pre-incubation method). The test item did not show mutagenic effects in the second experiment, either. The test item showed no precipitates on the plates in all tested concentrations. Signs of toxicity towards all bacteria strains could be observed in the two highest concentrations (1500 and 750 μg/plate). The sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

Under the conditions of the test, the test item did not show mutagenic effects towards Salmonella typhimurium, strains TA97a, TA98, TA100, TA102 and TA1535. Therefore, no concentration-effect relationship could be determined. The test item 2,4-di-tert-butylphenol is considered as “not mutagenic under the conditions of the test”.

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:
1998-05-19 to 1998-08-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Purity: 99.72%
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (lot #E05828-015), purchased from PAA Laboratories GmbH, Germany
- Properly maintained: yes, stock culture maintained under liquid nitrogen
- 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: cell cycle length approx. 16 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:
1 / 2 / 4 / 6 / 10 / 20 / 40 / 60 / 80 / 100 µg/ml in the first test
1 / 4 / 5 / 6 / 10 µg/ml in the second test
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 100 µg/ml (homogeneous emulsion).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
MEM medium containing 1% acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation Migrated to IUCLID6: 0.02 and 0.03 µg/ml
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation Migrated to IUCLID6: 2 and 3 µg/ml
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: 20 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 occurrence 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: unsoluble
- Precipitation: no precipitation up to 100 µg/ml
- Other confounding effects: none


RANGE-FINDING/SCREENING STUDIES: 10 different concentrations in the range of 1 - 100 µg/ml 2,4-di-tert Butylphenol were tested in the presence and absence of exogenous metabolic activation. On the basis of the determination of the mitotic index, the following concentrations were scored for chromosomal aberration induction: 0 / 1 / 6 / 10 µg/ml without metabolic activation in the first and second test; 0 / 1 / 4 / 6 µg/ml with metabolic activation in the first test, 0 / 1 / 4 / 5 / 6 µg/ml with metabolic activation in the second test.

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: First occurrence of cytotoxicity at 5 µg/ml in test #1, from 10 to 100 µg/ml no cells were on the slides. In test #2 cytotoxic effects were seen even at a concentration of 4 µg/ml, above 5 µg/ml test substance no cells were on the slides.
- Without metabolic activation: First occurrence of cytotoxicity at 6 µg/ml, in the range of 20 to 100 µg/ml no cells could be found on the slides.

GENOTOXIC EFFECTS:
- With metabolic activation: Treatment with the top concentration of 6 µg/ml 2,4-Di-tert-butylphenol resulted in an effect of very high statistical significance (P < 0.001) in test #1. The concentration of 4 µg/ml did not result in statistically or biologically significant increases in the frequency of cells with chromosome aberrations excluding gaps. In order to establish a dose response relationship, in the repeat experiment (test #2 with metabolic activation) an additional fourth test compound concentration of 5 µg/ml was introduced. Treatment with 5 µg/ml resulted in an effect of very high statistically significance (P < 0.001), 4 µg/ml 2,4-Di-tert-butylphenol showed no statistically significant effects in the aberration frequency in relation to the negative control. The determination of the mitotic index of test #2 showed a high cytotoxic effect in the concentration of 6 µg/ml 2,4-Di-tert-butylphenol, a scoring of aberrations was not possible. (See tables below).
- Without metabolic activation: In both experiments (#1 and #2) treatment with 2,4-Di-tert-butylphenol did not result in statistically or biologically significant increases in the frequency of cells with chromosome aberrations excluding gaps. (See tables below).
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.

Influence on the mitotic index:

test #1
without S9 mix  with S9 mix
concentration[µg/ml] absoluteMitotic index[%] relativeMitotic index[%]   concentration[µg/ml]  absoluteMitotic index[%]  relativeMitotic index[%]
 Neg. control  6.3 100  Neg. control  5.6   100
 1 5.6   88.9  1  4.7  84.7
 6  5.1  81.0  4  6.7  120.7
 10  4.4  69.0  6  2.5  44.1*

test #2
without S9 mix  with S9 mix
concentration[µg/ml] absoluteMitotic index[%] relativeMitotic index[%]   concentration[µg/ml]  absoluteMitotic index[%]  relativeMitotic index[%]
 Neg. control  14.7 100 Neg. control  6.3  100
 1 11.6  78.8  1  7.5  120.0
 6  6.3  43.0*  4  2.5  40.0*
 10  4.6  31.4*  5  1.4  22.4*

* relative mitotic indices lower than 50%

Genotoxic effects:

test #1

concentration[µg/ml]  S9 mix  Total # of mitotic cells scored  Proportion of cells with aberrations excl. gaps[%]  significance
 Neg. control - 200  0.0  -
 1  -  200  0.0  ns
 6  -  200  1.5  ns
 10  -  400  1.8  ns
 Pos. controlMMC 0.03  -  183  17.5  s
 Neg. control  +  200  1.0  -
 1  +  200  0.5  ns
 4  +  200  1.0  n
 6  +  200  9.5  ***
 Pos. control CP 3  200  9.5  s
 Pos. control CP 4  +  200  18.0  s

test #2

concentration[µg/ml]  S9 mix  Total # of mitotic cells scored  Proportion of cells with aberrations excl. gaps[%]  significance
 Neg. control - 200  0.0  -
 1  -  200  0.5  ns
 6  -  201  2.5  ns
 10  -  200  1.0  ns
 Pos. controlMMC 0.03  -  200  24.5  s
 Neg. control  +  200  0.5  -
 1  +  200  0.0  ns
 4  +  200  3.5  ns
 5  +  228  9.2  ***
 Pos. control CP 3  200  8.0  s
 Pos. control CP 4  +  200  12.0  s

ns = not significant s = significant, no statistical evaluation *** = statistically significant (P<0.001)

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

It is concluded, that in both experiments with metabolic activation, treatment with 2,4-di-tert-Butylphenol resulted in chromosomal aberration frequencies being significantly higher (biologically and statistically) than negative control values in the highest concentrations. A dose response relationship could not be established statistically in the frequency of cells with chromosomal aberrations excluding gaps. A connection between the chromosomal aberrations and the high cytotoxicity of the test substance can not be excluded.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
2,4-DTBP which is structurally similar to 2,6-DTBP
Reason / purpose:
read-across source
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested

Pre-experiment for toxicity

(the results were obtained from CCR Project 243628, Chromosome aberration)

Plating Efficiency Assay without metabolic activation, 495 single cells were seeded into each flask

Conc. Per ml

Colonies counted

mean

PE% relative

Flask I

Flask II

Negative control

301

248

274.5

 

Solvent control (DMSO)

280

271

275.5

100.0

0.1µg

266

291

278.5

101.1

1.0µg

221

259

240.0

87.1

3.0µg

288

300

294.0

106.7

6.0µg

2

9

5.5

2.0

10.0µg

0

0

0.0

0.0

30.0µg

0

0

0.0

0.0

50.0µg

0

0

0.0

0.0

100µg

0

0

0.0

0.0

Plating Efficiency Assay with metabolic activation, 495 single cells were seeded into each flask

onc. Per ml

Colonies counted

mean

PE% relative

Flask I

Flask II

Negative control

309

309

309.0

 

Solvent control (DMSO)

299

271

285.0

100.0

0.1µg

321

287

304.0

106.7

1.0µg

313

281

297.0

104.2

3.0µg

282

302

292.0

102.5

6.0µg

301

299

300.0

105.3

10.0µg

256

284

270.0

94.7

30.0µg

320

279

299.5

105.1

50.0µg

0

0

0.0

0.0

100µg

0

0

0.0

0.0

The test article was tested in experiment I (without S9 mix) and II (with S9 mix).

In the first experiment the plating efficiency was reduced at the highest concentration but in the second experiment no decrease of the plating efficiency was observed up to the highest concentration (see tables I and IV), PE% relative).

Table I: toxicity data, experiment I

Column

Conc. Per ml

S9 mix

Number of cells per flask*

PE%** absolute

PE%*** relative

seeded

found

mean

I/II

I

II

 

1

2

3

4

5

6

7

8

Negative control

0.00µg

-

497

298

299

298.5

60.1

100.0

Solvent control DMSO

0.00µg

-

497

260

260

260.0

52.3

100.0

 Positive control EMS

0.60µg

-

497

197

160

178.5

35.9

59.8

Test article

0.30µg

-

497

266

257

261.5

52.6

100.6

Test article

1.00µg

-

497

359

232

245.5

49.4

94.4

Test article

2.00µg

-

497

258

252

255.0

51.3

98.1

Test article

4.00µg

-

497

309

272

290.5

58.5

111.7

Test article

6.00µg

-

497

220

226

223.0

44.9

85.8

Test article

8.00 µg

-

497

13

20

16.5

3.3

6.3

Negative control

0.00µg

+

497

307

305

306.0

61.6

100.0

Solvent control DMSO

0.00µg

+

497

246

248

247.0

49.7

100.0

Positive control DMBA

3.85µg

+

497

270

249

259.5

52.2

105.1

Test article

3.00µg

+

497

256

271

263.5

53.0

86.1

Test article

10.00µg

+

497

227

234

230.5

46.4

75.3

Test article

20.00µg

+

497

253

253

253.0

50.9

82.7

Test article

30.00µg

+

497

244

259

251.5

50.6

82.2

Test article

40.00µg

+

497

258

299

278.5

56.0

91.0

Test article

50.00µg

+

497

217

240

228.5

46.0

74.7

*only colonies with more than 50 cells 7 days after seeding were scored

** PE absolute (value column 6/value column 3 x 100)

***PE relative (value column 6/value column 6 of corresponding control x 100)

Table II: Mutagenicity data, experiment I (part 1: cell survival)

Column

Conc. Per ml

S9 mix

Number of cells per flask*

Factor** calculated

Cells seeded

Cells*** survived

seeded

found

mean

 

 

 

 

I/II

I

II

 

1

2

3

4

5

6

7

8

9

Negative control

0.00µg

-

556

402

367

384.5

0.69

387000

267030

Negative control DMSO

0.00µg

-

474

391

432

411.5

0.87

453000

394110

 Positive control EMS

0.60µg

-

567

457

431

444.0

0.78

375000

292500

Test article

0.30µg

-

486

380

410

395.0

0.81

450000

364500

Test article

1.00µg

-

526

300

335

317.5

0.60

480000

288000

Test article

2.00µg

-

Culture was not continued

 

 

 

Test article

4.00µg

-

539

321

333

327.0

0.61

426000

259860

Test article

6.00µg

-

Culture was not continued

 

 

 

Test article

8.00 µg

-

530

335

363

349.0

0.66

372000

245520

Negative control

0.00µg

+

568

380

373

376.5

0.66

450000

297000

Negative control DMSO

0.00µg

+

537

357

344

350.5

0.65

432000

280800

 Positive control DMBA

3.85µg

+

479

307

323

315.0

0.66

423000

279180

Test article

3.00µg

+

579

374

331

352.5

0.61

402000

245220

Test article

10.00µg

+

461

324

330

327.0

0.71

450000

319500

Test article

20.00µg

+

Culture was not continued

 

 

Test article

30.00µg

+

497

316

315

315.5

0.63

435000

274050

Test article

40.00µg

+

Culture was not continued

 

 

 

Test article

50.00µg

+

486

264

297

280.5

0.58

435000

252300

*only colonies with more than 50 cells 7 days after seeding in normal medium were scored

** factor calculated (value column 6/value column 3)

***cells survived after plating in TG containing medium (value column 8 x value column 7)

Table III: Mutagenicity data, experiment I (part 2: mutation rates)

Column

Conc per ml

S9 mix

Number of mutant colonies per flask* found after plating in TG medium

Standard deviation

Mutant** colonies per 106cells

I

II

III

IV

V

mean

1

2

3

4

5

6

7

8

9

10

Negative control

0.00µg

-

7

1

11

6

4

5.8

3.7

21.7

Negative control DMSO

0.00µg

-

5

1

3

4

3

3.2

1.5

8.1

 Positive control EMS

0.60µg

-

103

83

93

91

80

90.0

9.1

307.7

Test article

0.30µg

-

14

16

18

11

8

13.4

4.0

36.8

Test article

1.00µg

-

3

6

7

4

10

6.0

2.7

20.8

Test article

2.00µg

-

Culture was not continued

 

 

 

 

Test article

4.00µg

-

4

8

8

6

9

7.0

2.0

26.9

Test article

6.00µg

-

Culture was not continued

 

 

 

 

Test article

8.00 µg

-

10

3

6

4

2

5.0

3.2

20.4

Negative control

0.00µg

+

5

8

5

8

11

7.4

2.5

24.9

Negative control DMSO

0.00µg

+

14

11

18

11

15

13.8

2.9

49.1

 Positive control DMBA

3.85µg

+

131

123

126

139

101

124.0

14.2

444.2

Test article

3.00µg

+

4

10

5

12

10

8.2

3.5

33.4

Test article

10.00µg

+

5

6

8

3

5

5.4

1.8

16.9

Test article

20.00µg

+

Culture was not continued

 

 

 

 

Test article

30.00µg

+

6

5

6

8

8

6.6

1.3

24.1

Test article

40.00µg

+

Culture was not continued

 

 

 

 

Test article

50.00µg

+

5

9

8

11

5

7.6

2.6

30.1

*only colonies with more than 50 cells 8 days after seeding in TG medium were scored

**value column 8 x106/value column 9 (table I)

Table IV: toxicity data, experiment II

Column

Conc. Per ml

S9 mix

Number of cells per flask*

PE%** absolute

PE%*** relative

seeded

found

mean

I/II

I

II

 

1

2

3

4

5

6

7

8

Negative control

0.00µg

-

497

295

296

295.5

59.5

100.0

Solvent control DMSO

0.00µg

-

497

289

262

275.5

55.4

100.0

 Positive control EMS

0.60µg

-

497

291

240

265.5

53.4

89.8

Test article

0.30µg

-

497

353

277

315.0

63.4

114.3

Test article

1.00µg

-

497

346

342

344.0

69.2

124.9

Test article

2.00µg

-

497

280

287

283.5

57.0

102.9

Test article

4.00µg

-

497

276

322

299.0

60.2

108.5

Test article

6.00µg

-

497

371

341

356.0

71.6

129.2

Test article

8.00 µg

-

497

310

315

312.5

62.9

113.4

Negative control

0.00µg

+

497

325

314

319.5

64.3

100.0

Solvent control DMSO

0.00µg

+

497

317

297

307.0

61.8

100.0

Positive control DMBA

3.85µg

+

497

191

206

198.5

39.9

64.7

Test article

3.00µg

+

497

303

284

293.5

59.1

91.9

Test article

10.00µg

+

497

315

267

291.0

58.6

91.1

Test article

20.00µg

+

497

299

297

298.0

60.0

93.3

Test article

30.00µg

+

497

289

303

296.0

59.6

92.6

Test article

40.00µg

+

497

282

272

277.0

55.7

86.7

Test article

50.00µg

+

497

263

294

278.5

56.0

87.2

*only colonies with more than 50 cells 7 days after seeding were scored

** PE absolute (value column 6/value column 3 x 100)

***PE relative (value column 6/value column 6 of corresponding control x 100)

Table V: mutagenicity data experiment II (part 1: cell survival)

Column

Conc. Per ml

S9 mix

Number of cells per flask*

Factor** calculated

Cells seeded

Cells*** survived

seeded

found

mean

 

 

 

 

I/II

I

II

 

1

2

3

4

5

6

7

8

9

Negative control

0.00µg

-

487

512

452

482.0

0.99

392000

388080

Negative control DMSO

0.00µg

-

450

367

420

393.5

0.87

435000

378450

 Positive control EMS

0.60µg

-

515

434

448

441.0

0.86

444000

381840

Test article

0.30µg

-

497

398

400

399.0

0.80

426000

340800

Test article

1.00µg

-

476

375

401

388.0

0.82

498000

408360

Test article

2.00µg

-

Culture was not continued

 

 

 

Test article

4.00µg

-

452

386

421

403.5

0.89

492000

437880

Test article

6.00µg

-

Culture was not continued

 

 

 

Test article

8.00 µg

-

480

446

460

453.0

0.94

435000

408900

Negative control

0.00µg

+

505

377

397

387.0

0.77

369000

284130

Negative control DMSO

0.00µg

+

492

338

436

387.0

0.79

306000

241740

 Positive control DMBA

3.85µg

+

494

333

334

333.5

0.68

396000

269280

Test article

3.00µg

+

495

369

393

381.0

0.77

402000

309540

Test article

10.00µg

+

510

346

308

327.0

0.64

378000

241920

Test article

20.00µg

+

Culture was not continued

 

 

Test article

30.00µg

+

533

245

283

264.0

0.50

387000

193500

Test article

40.00µg

+

Culture was not continued

 

 

 

Test article

50.00µg

+

493

407

367

387.0

0.78

384000

299520

*only colonies with more than 50 cells 7 days after seeding in normal medium were scored

** factor calculated (value column 6/value column 3)

***cells survived after plating in TG containing medium (value column 8 x value column 7)

See Table VI in overall remarks.

Taking into account the mutation rates found in the groups treated with the test article compared to the negative and solvent controls it can be concluded that no biologically relevant increase of point mutations was observed. The test article did not induce reproducible concentration-related increase in the mutant colony numbers. The mutant values of the groups treated with the test article were in the range of the negative controls.

In this study in both experiment (with and without S9 mix) the range of the negative controls was from 0.7 up to 49.1 mutants per 1000000 cells, the range of the groups treated with the test article was from 0.5 up to 36.8 mutants per 1000000 cells.

EMS (0.6 mg(ml) and DMBA (3.85 µg/ml) were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusions:
Interpretation of results (migrated information):
negative non mutagenic in this HGPRT assay

In conclusion can be stated that during the described mutagenicity test and under the experimental conditions reported the test article did not induce point mutations at the HGPRT locus in V79 cells.
Executive summary:

The study was performed to investigate the potential of 2,6 -bis(1,1 -dimethyethyl)-phenol to induce gene mutations at the HGPRT locus in V79 cells of the Chinese hamster in vitro.

The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation.

The test article was tested with the following concentrations:

Experiment I and II:

Without S9 mix: 0.3, 1 -0, 2.0, 4.0, 6.0*, and 8.0 µg/ml

With S9 mix: 3.0, 10.0, 20.0*, 30.0, 40.0, 50.0 µg/ml

* culture was not continued in experiment I and II

According to the pre-experiment for toxicity the concentration ranges were selected to yield concentration related toxic effects. In the first experiment the highest concentration produced a low level of survival and the survival at the lowest concentration was approximately in the range of the negative control.

At the highest investigated concentration no relevant increase in mutant colony numbers was obtained in two independent experiments.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies.

In conclusion it can be stated that during the described mutagenecity test and under the experimental conditions reported the test article did not induce point mutations at the HGPRT locus in V79 cells.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro

Four in vitro studies were conducted:

- Two Bacterial Reverse Mutation Assays (Ames test)

- In vitro chromosomal aberration assay

- Gene mutation assay in Chinese hamster V79 cells

In the Ames test (Andres I. (2015)), which was chosen as key study, the results were negative for mutagenicity in all tester strains. Cytotoxicity was observed with and without metabolic activation at concentrations of 750 µg/plate and higher. The second, supporting study (Scholz N. (1991)) showed also no mutagenicity, cytotoxicity occurred already at concentrations of 200 µg/plate and higher with and without metabolic activation.

The results of the in vitro chromosomal aberration assay (Krueger N. (1998)) concluded, that in both experiments with metabolic activation, treatment with 2,4-di-tert-butylphenol resulted in chromosomal aberration frequencies being significantly higher (biologically and statistically) than negative control values in the highest concentrations. A dose response relationship could not be established statistically in the frequency of cells with chromosomal aberrations excluding gaps. A connection between the chromosomal aberrations and the high cytotoxicity of the test substance cannot be excluded.

A third in vitro study was conducted to assess the gene mutation in Chinese hamster V79 cells (Müllerschön H. (1991)) with 2,6-bis(1,1-dimethylethyl)-phenol. The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. It was concluded that the test article did not induce point mutations at the HGPRT locus in V79 cells.

In vivo

An in vivo study was conducted using TK 12891/1 Micronucleus Test in Bone Marrow Cells of CD Rats: 0 h + 24 h Oral Dosing and 48 h Sampling Anon (2008). It concluded that the test substance did not induce micronuclei in bone marrow cells when tested to the maximum tolerated doses of 1000 mg/kg/day in male and 800 mg/kg/day in female CD rats using a 0 h + 24 h oral dosing and 48 h sampling regimen.


Short description of key information:
In vitro
Bacterial reverse mutation test (Andres I. (2015)) with 2,4-di-tert-butylphenol - negative for mutagenicity with and without metabolic activation.
In vitro chromosomal aberration assay (Krueger N. (1998)) with 2,4-di-tert-butylphenol - negative without metabolic activation, ambiguous at the highest concentrations with metabolic activation. No dose response relationship was present in the frequency of cells with chromosomal aberrations excluding gaps, however a connection between the chromosomal aberrations and the high cytotoxicity of the test substance could not be excluded.
In vitro gene mutation in Chinese hamster V79 cells (Müllerschön H. (1991)) with 2,6-bis(1,1-dimethylethyl)-phenol, a structurally similar compound - negative for mutagenicity with and without metabolic activation.
In vivo
Mammalian erythrocyte micronucleus test in CD rats (Anon (2008)) - negative for the induction of micronuclei in bone marrow cells.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Negative

There is no evidence for a mutagenic mode of action for 2,4 -di-tert-butylphenol in either in vitro or in vivo assays.