Ethanol, 2-amino-, reaction products with ammonia, by-products from, distn. Residues

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

January 6, 1987 - January 9, 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was performed according to methods similar to OECD471 but strain E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 was not included. Negative results were not confirmed by an independent repeat.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

- The study was performed according to methods similar to OECD471 but strain E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 is not included.
- Negative results were not confirmed by an independent repeat.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Trientine

EC Number:

203-950-6

EC Name:

Trientine

Cas Number:

112-24-3

Molecular formula:

C6H18N4

IUPAC Name:

N,N'-bis(2-aminoethyl)ethane-1,2-diamine

Details on test material:

Chemical Rame: Triethylenetetramine - Sample A
Chemical Synonyms: TETA
I. D a #/ : 36-ARB-31-8
BRRC Sample #: 49-424
BRRC Account R: 86-22-18120

Sample No.: 798-95-1
Purity 95.7%
9.71% TAEA
56.41% L-TETA
17.68% DAEP
11.88% PEEDA

0.125% AEEA
0.31% AE-TAEA
0.156% 4 unknowns
0.72% unknown
1.04% unknown
1.18% unknown
0.42% unknown
0.14% unknown

Other impurities < 0.1%

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

Additional strain / cell type characteristics:

other: r-factor (ampilicin resistance) (TA100 and TA98), uvrB-, rfa-

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: uvrB-, rfa-

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

without S9 0.03, 0.1, 0.3, 1 and 2 mg/plate
with S9 0.1, 0.3, 1, 3 and 5 mg/plate

Vehicle / solvent:

water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation);

DURATION
- Preincubation period: none
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplcate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

The spontaneous reversion for the solvent controls should be within this laboratory's historical range. The positive controls should demonstrate that the test systems are responsive with known mutagens. A test chemical is considered to be a bacterial mutagen if the number of revertant colonies is at least twice the solvent control for at least one dose level and there is evidence of a dose-related increase in the number of revertant colonies. If a test chemical produces a marginal or weak response that cannot be reproduced in a second test, the test result will be considered negative, If there is no evidence of a dose-related increase in the number of revertant colonies and the number of revertant colonies is not twice the solvent control, then the test chemical is not considered to be a bacterial mutagen.

Statistics:

none

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA100, TA98, TA1537, TA1538

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

increases in number of revertant colonies: TA98 6.6-fold, TA100 4.3-fold, TA1538 2.6-fold, TA1537 3-fold

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

2mg/plate in all strains except TA100

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

2mg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

maximun 5-fold increase

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium, other: TA1537, TA1538

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Range finding study:
In preliminary tests to determine cytotoxicity, ten concentrations of TETA-Sample B ranging from 0.01 to 98 mglplate were tested with and without the presence of an S9 metabolic activation system. Cytotoxicity was defined as either a reduction in the number of revertant colonies or an inhibition of growth of the background lawn. Dose levels ranging from 3.0 to 98 -/plate produced complete absence of growth of the background lawn in the test without S9. A lower dose of 1.0 mg/plate produced no evidence of cytotoxicity, allowing confluent growth of the background lawn. In addition the 1.0 mg/plate dose level produced a 4.3-fold increase in relative numbers of revertant colonies indicating that a biologically effective dose level had been attained. In the preliminary test performed with activation, dose levels ranging from 10 to 98 mg/plate produced absence of growth of the background lawn and a dose of 5 mg/plate produced cytotoxicity evident by sparse growth of the background lawn. In addition, this dose produced a 5.3-fold increase in revertant colonies while a lower dose of 1 and 3 mg/plate produced a 7.1-fold and 6.2-fold increase above control levels, repsectively.
Based on the results of these preliminary toxicity tests, 5 doses ranging from 0.03 to 2.0 mg/plate were tested without S9 and 5 doses ranging from 0.1 to 5 mg/plate were tested in the presence of S9 in definitive mutagenicity experiments using triplicate cultures at each dose level.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

TETA-Sample A was considered to be mutagenic in this vitro bacterial assay.

Executive summary:

Triethylenetetramine - Sample A (TETA-Sample A) was tested for potential mutagenic activity using the Salmonella/microsome bacterial mutagenicity assay (Ames test). Test doses for the Ames test were chosen from data obtained in a preliminary study with strain TA100. Tests without a rat liver S9 activation system indicated that a concentration of 3.0 mg/glate was cytotoxic and produced absence of growth of the bacterial lawn. A slightly lower dose of 1.0 mg/plate allowed confluent growth of the background lawn. In the test with S9, a dose of 5 mg/plate produced cytotoxicity evident by sparse growth of the bacterial lawn. Higher doses produced complete absence of the background lawn. Based on these results, five doses ranging from 0.03 to 2.0 mg/plate were tested in the definitive test without S9 and a slightly higher range of 0.1 to 5 mg/plate were tested in the presence of the S9 metabolic activation system, These concentrations were rested with five different strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537, and TA1538) using triplicate cultures at each dose level for each strain.

In the test without S9, dose-related mutagenic activity was observed with all five strains except TA1535. In tests performed in the presence of a

rat-liver S9 metabolic activation system, strains TA98, TA100 and TA1535 had highly positive and dose related increases in numbers of revertant colonies. Thus, TETA-Sample A was considered to be mutagenic in this in vitro bacterial assay.