Ethanol, 2-amino-, reaction products with ammonia, by-products from

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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Glp guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ethanol, 2-amino-, reaction products with ammonia, by-products from

EC Number:

272-729-4

EC Name:

Ethanol, 2-amino-, reaction products with ammonia, by-products from

Cas Number:

68910-05-4

Molecular formula:

Unspecified

IUPAC Name:

Ethanol, 2-amino-, reaction products with ammonia, by-products from

Details on test material:

- Name of test material (as cited in study report):Diethylentriamine R
- Physical state: liquid
- Analytical purity: The identity of the test substance was confirmed...........

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-mix prepared form sprague Dawlay rat livers after Aroclor 1254 activation

Test concentrations with justification for top dose:

0, 20, 100, 500, 2500, 5000 µg/plate (SPT)

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: see details

Details on test system and experimental conditions:

Standard Plate Test
Salmonella typhimurium

Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] 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 about 42 - 45°C, and the remaining components are added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (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 purified water
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
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] 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 about 42 - 45°C, and the remaining components are added in the following order:

0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
Or
0.5 mL phosphate buffer (without metabolic activation)

After mixing, the samples are poured onto minimal agar plates within approx. 30 seconds.
The composition of the minimal agar (SA1 selective agar) is based on the description of Green, M.H.L. and Muriel, W.J. ( 5), with the exception of the amino acid 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.
In each experiment 3 test plates per dose or per control were used.

Positive controls:
2-Aminoanthracene: 2.5 µg/plate for strains: TA 1535, TA 100, TA 1537, TA 98 ( with S9 mix)
60 µg/plate for strain: E. Coli WP2 uvrA ( with S9 mix)
N-methyl-N`-nito-N-nitrosoguanidine : 5 µg/plate for strains: TA 1535, TA 100 (without S9 mix)
4-nitro-o-phenylenediamine: 10 µg/plate for strain: TA 98 (without S9 mix)
9-aminoacridine: 100 µg/plate for strain: TA 1537 (without S9 mix)
4-nitroquinoline-N-oxide: 5 µg/plate for strain: E.coli WP2 uvrA (without S9 mix)
Dissolved in DMSO.

The titer is generally determined only in the experimental parts with S9 mix both for the negative controls and for the two highest doses in all experiments.

Evaluation criteria:

Positive results:
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 S9 mix or after adding a metabolizing system.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Mutagenicity

Strain	Metabolic activation	Experiment No.	Concentration [µg/plate]	Replicates	Max. factor of revertants
TA 1535	No No	1 2	20-5000 20-5000	3 3	No relevant increase in the numbers of his+ or trp+ revertans
TA 100	No No	1 2	20-5000 20-5000	3 3	4.2 4.3
TA 1537	No No	1 2	20-5000 20-5000	3 3	2.4 2.3
TA 98	No No	1 2	20-5000 20-5000	3 3	3.0 3.5
E.coli WP2 uvrA	No No	1 2	20-5000 20-5000	3 3	8.7 9.6
TA 1535	Yes Yes	1 2	20-5000 20-5000	3 3	No relevant increase in the numbers of his+ or trp+ revertans
TA 100	Yes Yes	1 2	20-5000 20-5000	3 3	2.8 4.0
TA 1537	Yes Yes	1 2	20-5000 20-5000	3 3	1.6 2.5
TA 98	Yes Yes	1 2	20-5000 20-5000	3 3	2.7 3.4
E.coli WP2 uvrA	Yes Yes	1 2	20-5000 20-5000	3 3	4.0 5.7

Applicant's summary and conclusion

Conclusions:

Thus, under the experimental conditions chosen here, it is concluded that Diethylen-triamine R is a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.