6-(dibutylamino)-1,3,5-triazine-2,4(1H,3H)-dithione

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Species / strain / cell type:

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

Metabolic activation:

with and without

Test concentrations with justification for top dose:

The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment (see chapter 12.1.1 Pre-Experiment). 5000 µg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. Two independent experiments were performed with the following concentrations:
31.6, 100, 316, 1000, 2500 and 5000 µg/plate
As the results of the pre-experiment were in accordance with the criteria described above, these were reported as a part of the main experiment I.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

10.4.1. Bacteria
Five strains of S. typhimurium with the following characteristics were used:
TA 98:
his D 3052; rfa-; uvrB-; R-factor: frame shift mutations
TA 100:
his G 46; rfa-; uvrB-; R-factor: base-pair substitutions
TA 1535:
his G 46; rfa-; uvrB-: base-pair substitutions
TA 1537:
his C 3076; rfa-; uvrB-: frame shift mutations
TA 102:
his G 428 (pAQ1); rfa-; R-factor: base-pair substitutions
Tester strains TA 98, TA 1535 and TA 102 were obtained from MOLTOX, INC., NC 28607, USA. Tester strains TA 100 and TA 1537 were obtained from Xenometrix AG, Switzerland. They were stored as stock cultures in ampoules with nutrient broth (OXOID) supplemented with DMSO (approx. 8% v/v) over liquid nitrogen.
All Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium. They contain the deep rough (rfa) mutation, which deletes the polysaccharide side chain of the lipopolysaccharides of the bacterial cell surface. This increases cell permeability of larger substances. The other mutation is a deletion of the uvrB gene coding for a protein of the DNA nucleotide excision repair system resulting in an increased sensitivity in detecting many mutagens. This deletion also includes the nitrate reductase (chl) and biotin (bio) genes (bacteria require biotin for growth).
The tester strains TA 98, TA 100 and TA 102 contain the R-factor plasmid, pkM101. These strains are reverted by a number of mutagens that are detected weakly or not at all with the non R-factor parent strains. pkM101 increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA repair system which is normally present in these organisms [12], [15].
The properties of the S. typhimurium strains with regard to membrane permeability, ampicillin- and tetracycline-resistance as well as normal spontaneous mutation rates are checked regularly according to Ames et al. [7]. In this way it is ensured that the experimental conditions set up by Ames are fulfilled.
10.4.2. Preparation of Bacteria
Samples of each tester strain were grown by culturing for 12 h at 37 °C in Nutrient Broth to the late exponential or early stationary phase of growth (approx. 109 cells/mL). The nutrient medium consists per litre:
8 g Nutrient Broth
5 g NaCl
A solution of 125 µL ampicillin (10 mg/mL) (TA 98, TA 100, TA 102) was added in order to retain the phenotypic characteristics of the strain.
10.4.3. Agar Plates
The Vogel-Bonner Medium E agar plates with 2% glucose used in the Ames Test were prepared by Eurofins Munich. Quality controls were performed.
Vogel-Bonner-salts contain per litre:
10 g MgSO4 x 7 H2O
100 g citric acid
175 g NaNH4HPO4 x 4 H2O
500 g K2HPO4
Sterilisation was performed for 20 min at 121 °C in an autoclave.
Vogel-Bonner Medium E agar plates contain per litre:
15 g Agar Agar
20 mL Vogel-Bonner salts
50 mL glucose-solution (40%)
Sterilisation was performed for 20 min at 121 °C in an autoclave.
10.4.4. Overlay Agar
The overlay agar contains per litre:
7.0 g Agar Agar
6.0 g NaCl
10.5 mg L-histidine x HCl x H2O
12.2 mg biotin
Sterilisation was performed for 20 min at 121 °C in an autoclave.
10.4.5. Mammalian Microsomal Fraction S9 Mix
The bacteria most commonly used in these reverse mutation assays do not possess the enzyme system which, in mammals, is known to convert promutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous metabolic system was added in the form of mammalian microsome enzyme activation mixture.
10.4.6. S9 Homogenate
The S9 liver microsomal fraction was prepared at Eurofins Munich and obtained from Trinova Biochem GmbH, Gie?en, Germany. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route (Eurofins Munich) and male Sprague Dawley rats were induced with phenobarbital / β-naphthoflavone (Trinova).
The following quality control determinations were performed by Eurofins Munich:
a) Biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene
b) Sterility Test
A stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4 mL and stored at -75 °C.
The protein concentration in the S9 preparation (Lot: 020617) was 34.2 mg/mL.
The following quality control determinations were performed by Trinova Biochem GmbH:
a) Alkoxyresorufin-0-dealkylase activities
b) Test for the presence of adventitious agents
c) Promutagen activation (including biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene)
A stock of the supernatant containing the microsomes is frozen in aliquots of 5 mL and stored at  -75 °C.
The protein concentration in the S9 preparation (Lot: 3727) was 33.4 mg/mL.
10.4.7. Preparation of S9 Mix
The S9 mix preparation was performed according to Ames et al. [7].
100 mM of sodium-ortho-phosphate-buffer, pH 7.4, was ice-cold added to the following pre-weighed sterilised reagents to give final concentrations in the S9 mix of:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
This solution was mixed with the liver 9000 x g supernatant fluid in the following proportion:
co-factor solution 9.5 parts
liver preparation 0.5 parts
During the experiment the S9 mix was stored on ice.
 
10.4.8. S9 Mix Substitution Buffer
The S9 mix substitution buffer was used in the study as a replacement for S9 mix, without metabolic activation (-S9).
Phosphate-buffer (0.2 M) contains per litre:
0.2 M NaH2PO4 x H2O 120 mL
0.2 M Na2HPO4 880 mL
The two solutions were mixed and the pH was adjusted to 7.4. Sterilisation was performed for 20 min at 121 °C in an autoclave.
This 0.2 M phosphate-buffer was mixed with 0.15 M KCl solution (sterile) in the following proportion:
0.2 M phosphate-buffer 9.5 parts
0.15 M KCl solution 0.5 parts
This S9 mix substitution buffer was stored at 4 °C.

Evaluation criteria:

A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100, TA 102)
- the negative control plates (A. dest.) with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range (2014 -2016)):

- S9 + S9
min max min max
TA 98 11 58 15 59
TA 100 49 155 62 160
TA 1535 4 41 3 38
TA 1537 3 35 3 36
TA 102 141 472 157 586
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, 6-(dibutylamino)-1,3,5-triazine-2,4(1H,3H)-dithione did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, 6-(dibutylamino)-1,3,5-triazine-2,4(1H,3H)-dithione is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

In order to investigate the potential of 6-(dibutylamino)-1,3,5-triazine-2,4(1H,3H)-dithione for its ability to induce gene mutations the plate incorporation test (experiment I) and the pre-incubation test (experiment II) were performed with theSalmonella typhimuriumstrains TA 98, TA 100, TA 1535, TA 1537 and TA 102.

In two independent experiments several concentrations of the test item were used. Each assay was conductedwithandwithoutmetabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

31.6, 100, 316, 1000, 2500 and 5000 µg/plate

No precipitation of the test item was observed in any tester strain used in experiment I and II (withandwithoutmetabolic activation).

No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (withandwithoutmetabolic activation) in experiment I and II with one exception: Toxic effects of the test item were noted in experiment I in tester strain TA 100 at a concentration of 5000 µg/plate (withoutmetabolic activation).

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with 6-(dibutylamino)-1,3,5-triazine-2,4(1H,3H)-dithione at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

All criteria of validity were met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

6-(dibutylamino)-1,3,5-triazine-2,4(1H,3H)-dithione is considered to be non-mutagenic in this bacterial reverse mutation assay.