Tetrakis(2-hydroxyethyl)ammonium hydroxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tetrakis(2-hydroxyethyl)ammonium hydroxide

EC Number:

211-158-7

EC Name:

Tetrakis(2-hydroxyethyl)ammonium hydroxide

Cas Number:

631-41-4

Molecular formula:

C8H20NO4.HO

IUPAC Name:

tetrakis(2-hydroxyethyl)azanium hydroxide

Test material form:

liquid

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from male Wistar rats (Acroclor 1254)

Test concentrations with justification for top dose:

1st experiment: 0; 100, 500; 2500; 12500 and 25000 µg/plate (standard plate test, with and without S9-mix)
2nd experiment: 0; 1250, 2500; 5000; 10000; 20000 µg/plate (preincubation test, with and without S9-mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: good solubility in water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene, N-methyl-N`-nitro-N-nitrosoguanidine, 4-nitro-o-phenylendiamine,

Details on test system and experimental conditions:

Standard plate test
Salmonella typhimurium
The experimental procedure of the standard plate test (plate incorporation method) is based an the method on Ames et al. (1, 2). Test tubes containing 2-ml portions of soft agar (overlay 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 or vehicle
0.1 ml fresh bacterial culture
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. 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 - 72 hours in the dark, the bacterial colonies (his revertants) are counted.

Escherichia coli
The experimental procedure is based on the method of Ames et al. (1, 2). Test tubes containing 2-ml portions of soft agar (overlay 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 or vehicle
0.1 ml fresh bacterial culture
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 minimal agar plates within approx. 30 seconds.
Composition of the minimal agar:
The composition of the minimal agar (SA1 selective agar) is based an the description af Green, M.H.L. and Muriel, W.J. (5), with the exception of solution E (tryptophan solution), which has previously been added to the soft agar:
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 at 37°C for 48 - 72 hours in the dark, the bacterial colonies (trp+ revertants) are counted.

Preineubation test
The experimental procedure is based an the method described by Yahagi et ah. (7) and Matsushima et ah. (8).
0.1 ml test solution or vehicle, 0.1 ml bacterial suspension and 0.5 ml S-9 mix are incubated at 37°C for the duratian of about 20 minutes using a shaker. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met:
A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.

A test substance is generally considered nonmutagenic in this test if:
The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TOXICITY
A bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed depending on the strain and test conditions from about 12,500 µg/plate onward (SPT) or from about 5,000 µg - 10,000 µg/plate onward (PIT).

No test substance precipitation was found.

An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9-mis or after the addition of a metabolizing system.

Applicant's summary and conclusion

Conclusions:

According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimantal conditions chosen here.