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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2,4-di-tert-butylphenol
EC Number:
202-532-0
EC Name:
2,4-di-tert-butylphenol
Cas Number:
96-76-4
Molecular formula:
C14H22O
IUPAC Name:
2,4-di-tert-butylphenol
Test material form:
solid
Specific details on test material used for the study:
Batch-no.: 1437
Purity: min. 99%

Method

Species / strainopen allclose all
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.
Controlsopen allclose all
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

Results and discussion

Test resultsopen allclose all
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.

Any other information on results incl. tables

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)

Applicant's summary and conclusion

Conclusions:
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”.