4-tert-butylbenzaldehyde

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions (missing strain for detecting cross-link mutations, e.g. TA102 or E. coli WP2)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

; only 4 S. typhimurium strains were used (TA1535, TA100, TA1537 and TA98), 5 are recommended in OECD 471

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-tert-butylbenzaldehyde

EC Number:

213-367-9

EC Name:

4-tert-butylbenzaldehyde

Cas Number:

939-97-9

Molecular formula:

C11H14O

IUPAC Name:

4-tert-butylbenzaldehyde

Details on test material:

- Name of test material (as cited in study report): 4-tertiaer-Butylbenzaldehyde (TBA)
- Physical state: colourless to yellowish liquid
- Analytical purity: 96.0 %
- Storage condition of test material: +4 °C to +6 °C (under N2)

Method

Target gene:

Determination of the rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his+).

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium strains TA 1535, TA 100, TA 1537, TA 98

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from the liver of Aroclor 1254 treated male Sprague-Dawley rats

Test concentrations with justification for top dose:

1st Experiment:
Strains: TA 100, TA 98
Doses : 0, 20 , 100, 500, 2500 and 5000 µg/plate

2nd Experiment :
Strains : TA 1535, TA 100, TA 1537, TA 98
Doses : 0, 4, 20, 100, 500 and 1000 µg/plate

3rd Experiment :
Strains : TA1535, TA100, TA1537, TA98
Doses: PIT ; all tester strains:
0, 0.8, 4, 20, 100 and 500 µg/plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see "Details on test system and conditions"

Details on test system and experimental conditions:

METHOD OF APPLICATION:
A standard plate test and a preincubation test were conducted, both with and without metabolic activation (S9 mix). Each test was conducted in triplicates.

STANDARD PLATE TEST:
The experimental procedure is based on the method of Ames et al. (Mut. Res. 31: 347-364, 1975):
Test tubes containing 2 ml portions of soft agar which consists of 100 ml agar (0.6% agar + 0.6% NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45°C and the remaining components are added in the following order:
0.1 ml test solution
0.1 ml bacterial suspension
0.5 ml S-9 mix (in tests with metabolic activation)
or
0.5 ml phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.

PREINCUBATION TEST:
The experimental procedure is based on the method described by Yahagi et al. (Mut. Res. 48: 121-130, 1977) and Matsushima et al. (Factors modulating mutagenicity in microbial tests. In: Norpoth, K.H. and R.C. Garner, Short-Term Test Systems for Detecting Carcinogens. Springer Verlag Berlin, Heidelberg, New York, 1980):
0.1 ml test solution, 0.1 ml bacterial suspension and 0.5 ml S-9 mix are incubated at 37°C for the duration of 20 minutes. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the Vogel-Bonner agar plates within approx. 30 seconds.
Composition of the minimal glucose agar:
980 ml aqua dest.
20 ml Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose monohydrate.
After incubation at 37 °C for 48 hours in the dark, the bacterial colonies (his+ revertants) are counted.

CONTROLS:
- Negative control:
Parallel with each experiment with and without S-9 mix, a negative control (with vehicle DMSO) is carried out for each tester strain in order to determine the spontaneous mutation rate.
- Positive controls:
with S-9 mix: - 10 µg/plate 2-aminoanthracene (2-AA, dissolved in DMSO) for the strains TA 100, TA 98, TA 1537 and TA 1535
without S-9 mix: - 5 µg/plate N-methyl-N´-nitro-N-nitrosoguanidine (MNNG, dissolved in DMSO) for the strains TA 100 and TA 1535 - 10 µg/plate 4-nitro-o-phenylendiamine (NPD, dissolved in DMSO) for the strain TA 98 - 100 µg/plate 9-aminoacridine (AAC, dissolved in DMSO) for the strain TA 1537

TITER DETERMINATION:
In general, the titer is determined only in the experiments with S-9 mix both without test substance (solvent only) and after adding the two highest amounts of substance. For this purpose, 0.1 ml of the overnight cultures is diluted to 1 x 10exp-6 in each case. Test tubes containing 2 ml portions of soft agar containing maximal amino acid solution (5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45 °C, and the remaining components are added in the following order:
0.1 ml solvent (without and with test substance)
0.1 ml bacterial suspension (dilution: 1 x 10exp-6)
0.5 ml S-9 mix
After mixing, the samples are poured onto the Vogel-Bonner agar plates within approx. 30 seconds. After incubation at 37°C for 48 hours in the dark, the bacterial colonies are counted.

Evaluation criteria:

In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: A bacteriotoxic effect was observed depending on the strain and test conditions at doses >= 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A bacteriotoxic effect (reduced his background growth, decrease in the number of his+ revertants was observed depending on the strain and test cond itions at doses >= 500 µg/plate .

Remarks on result:

other: strain/cell type: Salmonella typhimurium strains TA 1535, TA 100, TA 1537, TA 98

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Mutagenicity data: 

Standard Plate Test 1
Maximum number of his+ revertants/plate (mean +/- SD):
strain      control         treatment (conc. in µg/plate)
          -S9      +S9       -S9             +S9
---------------------------------------------------------
TA100    126+/-7  118+/-10  126+/-12 (20)    146+/-12 (20)
TA98     25+/-2   41+/-2    24+/-3 (500)     39+/-5 (20)

Standard Plate Test 2
Maximum number of his+ revertants/plate (mean +/- SD):
strain      control         treatment (conc. in µg/plate)
          -S9      +S9       -S9             +S9
---------------------------------------------------------
TA100    106+/-6  129+/-3   135+/-5 (100)   143+/-10 (4)
TA98     22+/-3   38+/-4    27+/-2 (4)      39+/-4 (4)
TA1535   14+/-2   16+/-6    17+/-3 (500)    17+/-4 (100)
TA1537   11+/-7   12+/-2    13+/-3 (20)     12+/-3 (100)

Preincubation Test
Maximum number of his+ revertants/plate (mean +/- SD):
strain      control         treatment (conc. in µg/plate)
          -S9      +S9       -S9             +S9
---------------------------------------------------------
TA100    111+/-6  105+/-5   119+/-12 (100)  136+/-7 (100)
TA98     31+/-1   35+/-5    29+/-4 (4)      39+/-2 (0.8)
TA1535   17+/-3   15+/-2    22+/-3 (100)    18+/-4 (20)
TA1537    9+/-2   11+/-1    14+/-4 (0.8)    10+/-2 (4)


The positive controls showed the expected increase in his+ revertants/plate.

Data for the pos. controls: his+ revertants/plate (mean +/- SD):
Standard Plate Test 1 
strain     pos. control      
           -S9        +S9          
---------------------------------
TA100   1523+/-84  1723+/-204    
TA98    1243+/-140 1607+/-108  

Standard Plate Test 2          Preincubation Test
strain     pos. control             pos. control        
           -S9        +S9         -S9        +S9      
------------------------------------------------------
TA100   1197+/-127 1105+/-85    675+/-10  1133+/-341 
TA98     794+/-35  1132+/-133  1197+/-120 1437+/-59 
TA1535  1260+/-10   200+/-67    814+/-49   170+/-33
TA1537   464+/-95   265+/-34   1197+/-55   116+/-9