Tetrahydrothiopyran-3-carboxaldehyde

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: non-GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test substance: TPA
- Chemical name: 3,4,5,6-tetrahydro-2H-thiopyrane-3-aldehyde
- Batch No.: LJ 1-7718-126
- Test substance No.: 85/249
- Degree of purity: 97.44%
- Appearance, consistency: colorless liquid
- Storage: +4° C

The stability of the test substance throughout the study period will be verified by reanalysis at a later date.

The stability of the test substance in the solvent DMS0 was determined analytically.

Method

Target gene:

histidine operon

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 3

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix (Aroclor 1254 induced)

Test concentrations with justification for top dose:

1st Experiment: Doses: 0, 20, 100, 500, 2500 and 5000 µg/plate (with and without S-9 mix)
2nd Experiment: Doses: 0, 20, 100, 500, 1500 and 2500µg (without S-9 mix), 0, 20, 100, 500, 2500 and 5000 µg (with S-9 mix)
3rd, 4th and 5th Experiments: Strain: Doses: 0, 20, 100, 500, 2500 and 5000 µg/plate (with and without S-9 mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see details below

Details on test system and experimental conditions:

INTRODUCTION
The Ames test and E. coli - reverse mutation assay are short-term tests in bacteria and are used as screening methods for detecting a paint mutagenic effect of chemical substances. Since most of the substances are not mutagenic or carcinogenic themselves, but only after metabolic transformation, and since the main part of all metabolic processes is catalyzed by the enzyme systems of the liver, the tests are carried out not only directly, but also in the presence of
a metabolizing system obtained from rat livers. For this purpose, rats are pretreated with Aroclor 1254 for an activation of the enzymes which metabolize foreign substances.

S-9 fraction
The S-9 fraction is prepared according to Ames et al.
5 male Sprague-Dawley rats (200 - 300 g) receive a single intraperitoneal injection of 500 mg Aroclor 1254 (as a 20% solution in peanut oil – w/v) per kg body weight 5 days before sacrifice. During this time the animals are housed in Makrolon cages in air-conditioned rooms. The day/night rhythm is 12 hours
S-9 mix
The S-9 mix is prepared freshly prior to each experiment. For this purpose, a sufficient amount of S-9 fraction is thawed at room temperature and 3 volumes of S-9 fraction are mixed with 7 volumes of S-9 supplement (cofactors). This preparation the so-called S-9 mix, is kept on ice until used. The concentrations of the cofactors in the S-9 mix are:
MgCl 28 mM
KCl 33 mM
glucose-6-phosphate 5 mM
NADP 4 mM
phosphate buffer (pH 7.4) 100 mM
The phosphate buffer is prepared by mixing an Na 2HPO4 solution (25.42 g/l) with an NaH2PO4 solution (22.28 g/l) in a ratio of about 4 :1.

Bacteria
The rate of induced back mutations of several bacteria mutants from histidine auxotrophy to histidine prototrophy im determined. The indicator organisms TA 1535, TA 1537, TA 98 and TA 100 selected by Ames especially for this purpose are derivatives of Salmonella typhimurium. All strains have a defective excision repair system (uvrB), which prevents the repair of lesions which are induced in the DNA. and this deficiency results in greatly enhanced sensitivity of same mutagens. Furthermore, all strains show a considerably reduced hydrophilic polysaccharide layer (rfa), which leads to an increase in permeability to lipophilic substances.

The strains TA 1535 and TA 100 are derived from histidine-prototrophic Salmonella strains by the substitution mutation his G 46 and are used to detect base pair substitutions. TA 1537 and TA 98 are strains for the detection of frameshift mutagens. These strains carry different frameshift markers, i.e. the +1 mutant his C 3076 in the case of TA 1537 and the +2 type his D 3052 in the case of TA 98. The strains TA 98 and TA 100 carry an R factor plasmid pKM 101 and, in addition to having genes resistant to antibiotics, they have a modified post-replication DNA repair system, which increases the mutation rate by inducing a defective repair in the DNA; this again leads to a considerable increase in sensitivity. Escherichia coli WP2 uvrA is a derivative of E. coli WP2 with a deficient excision repair and is used to detect substances which induce base pair substitutions. The rate of induced back mutations from tryptophan auxotrophy to tryptophan independence is determined.

For testing. deep-frozen (-70° C to -80° C) bacterial cultures (1 ml in 15 ml glass tubes) are thawed at room temperature, 0.1 ml of this bacterial suspension is inoculated in nutrient broth solution (8 g Difco nutrient broth + 5 g NaCl/liter) and incubated in the shaking water bath at 37° C for 16 hours. As a rule, a germ density of >10e8 bacteria/ml is reached. These cultures grown overnight are kept in iced water from the beginning of the experiment until the end in order to prevent further growth.

Mutagenicity tests
Salmonella typhimurium
The experimental procedure is based an the method of Ames et al.
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 the Vogel-Bonner agar plates (minimal glucose 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 for 48 hours at 37° C in the dark, the bacterial colonies (his+ revertants) are counted.


Escherichia coli
The experimental procedure is based on the method of Ames et al.
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 tryptophan) 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 the agar plates within approx. 30 seconds.

Composition of the minimal agar:
The composition of the minimal agar (SAl selective agar) is based on the description of Green, M.H.L. and Muriel, W.J., with the exception of solution E (tryptophan solution), which has been added to the soft agar before:
300 ml solution B (agar)
100 ml solution A (saline solution)
8 ml solution C (glucose solution)
10 ml solution D (casein solution)
After incubation for 48 hours at 37° C in the dark, the bacterial colonies (trp+ revertants) are counted.

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 10e-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
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.

Checking out the tester strains
The Salmonella strains are checked for the following characteristics at regular intervals: deep rough character (rfa); UV sensitivity (delta uvrB); ampicillin resistance (R factor plasmid). Histidine auxotrophy is automatically checked in each experiment via the spontaneous rate.

Controls
Negative control
Parallel with each experiment with and without S-9 mix, a negative control (solvent control, sterility control) is carried out for each tester strain in order to determine the spontaneous mutation rate.

Positive control
The following positive control substances are used to check the mutability of the bacteria and the activity of the S-9 mix:

with S-9 mix:
10 µg 2-aminoanthracene (dissolved in DMSO)
for the strains TA 100, TA 98, TA 1537 and TA 1535

60 µg 2-aminoanthracene for E. coli WP2 uvrA

without S-9 mix:
5 µg N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) (dissolved in DMSO)
for the strains TA 100 and TA 1535

10 µg 4-nitro-o-phenylenediamine (dissolved in DMSO)
for the strain and TA 98

100 µg 9-aminoacridine chloride monohydrate (dissolved in DMSO)
for the strain TA 1537

10 µg N-ethyl-N'-nitro-N-nitroso-guanidine (ENNG) (dissolved in DMSO)
for the strain E. coli WP2 uvrA.

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.

Tester strains, doses, number of plates
1st Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98
Doses: 0, 20, 100, 500, 2500 and 5000 µg/plate
Solvent: DMSO
Type of test, standard plate test with and without test condition: S-9 Mix
Number of plates: 3 test plates per dose or per control

2nd Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98
Doses: 0, 20, 100, 500, 1500 and 2500µg (without S-9 mix)
0, 20, 100, 500, 2500 and 5000 µg (with S-9 mix)
Solvent: DMS0
Type of test, standard plate test with and without test condition: S-9 Mix
Number of plates: 3 test plates per dose or per control

3rd, 4th and 5th Experiments
Strain: E. coli WP2 uvrA
Doses: 0, 20, 100, 500, 2500 and 5000 µg/plate
Solvent: DMSO
Type of test, standard plate test with and without test condition: S-9 mix
Number of plates: 3 test plates per dose or per control

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

Test results 1

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: only occasionally at the limit test concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS

Mutagenicity tests
Tests without S-9 mix
TA 1535; TA 100; TA 1537; TA 98; E. coli WP2 uvrA: No increase in the number of his+ and trp+ revertants

Tests with S-9 mix
TA 1535; TA 100; TA 1537; TA 98; E. coli WP2 uvrA: No increase in the number of his+ and trp+ revertants

Toxicity
A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ revertants) was observed from about 1 500 µg/plate onward (without S-9 mix) or at 5000 µg/plate (with S-9 mix). Using E. coli WP2 uvrA, only occasionally was a slight decrease in the number of trp+ revertants found at 5000 µg/plate.

Solubility
Complete solubility of the test substance in DMSO.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

SUMMARY

The substance TPA was tested for mutagenicity in the Ames test and in the Escherichia coli - reverse mutation assay.

 

Strains:

TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

 

Dose range:

20 µg - 5000 µg/plate

 

Test conditions:

Standard plate test without and with metabolic activation (S-9 mix)

 

Solubility:

Complete solubility of the test substance in DMSO

 

Toxicity:

With the Salmonella strains a bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ revertants) was observed from about 1500 µg/plate onward (without S-9 mix) and at 5000 µg/plate (with S-9 mix). Using E. coli WP2 uvrA a slight decrease in the number of trp+ revertants was only occasionally found at 5000 µg/plate.

 

Mutagenicity:

An increase in the number of his+ and trp+ revertants could not be observed either without S-9 mix or after the addition of a metabolizing system.

 

Assessment:

According to the results of the present study, the test substance TPA is not mutagenic in the Ames test and in the E. coli - reverse mutation assay under the experimental conditions chosen here.