Bis(2-hydroxyethyl)ammonium acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No studies are available on the genetic toxicity of DEA acetate. However, the mutagenicity and clastogenicity of structurally-related read-across compounds have been assessed in reliable mammalian and bacterial cell cultures. No evidence of mutagenicity or clastogenicity was observed.

Link to relevant study records

Reference

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:

weight of evidence

Study period:

2010-02-22 to 2010-04-26

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted according to Guidelines in a GLP certified laboratory on a read-across compound.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Genes in histidine operon

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes were routinely prepared from adult male Wister rats.

Test concentrations with justification for top dose:

Dose range finding concentrations: 100 to 5000 μg/plate.
First mutation assay concentration: 100 to 5000 μg/plate.
Independent repeat assay concentration: 100 to 5000 μg/plate.

Vehicle / solvent:

Milli-Q water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Milli-Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9: TA1535 - sodium azide (in Saline); TA1537 - 9-aminoacridine (in saline); TA98 - 2-nitrofluorene (in Milli-Q water); TA100 - methylmethanesulfonate (in DMSO); WP2uvrA - 4-nitroquinoline-N-oxide (in DMSO). With S9: 2-aminoanthracene

Details on test system and experimental conditions:

Test System: Salmonella typhimurium and Escherichia coli bacteria.

The Salmonella typhimurium strains were regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.

The Escherichia coli WP2uvrA was regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.

Stock cultures of the five strains were stored in liquid nitrogen (-196 deg C).

Evaluation criteria:

No formal hypothesis testing was done.

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might have entered into the final evaluation decision.

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Dose Finding Test

Triethanol amineacetate was tested in the tester strains TA100 and WP₂uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330, 5000 μg/plate in the absence and presence of S9-mix. This dose range finding test is reported as part of the first experiment of the mutation test (Table 1).

 

Precipitation of Triethanol amineacetate on the plates was not observed at the start or at the end of the incubation period in both tester strains.

 

No reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

 

In the dose range finding test, no increase in the number of revertants was observed upon treatment with Triethanol amineacetate under all conditions tested.

 

Mutation Assay

Based on the results of the dose range finding test, Triethanol amineacetate was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in two mutation assays. The first mutation experiment was performed with the strains TA1535, TA1537 and TA98 and the second mutation experiment was performed with the strains TA1535, TA1537, TA98, TA100 and WP₂uvrA. The results are shown in Table 1 and 2.

 

Precipitation of Triethanol amineacetate on plates was not observed at the start or at the ends of the incubation period.

 

In both mutation assays, there was no reduction of the bacterial background lawn and no decrease in the number of revertants at any of the concentration tested in all tester strains in the absence and presence of S9-mix, except for a slight reduction of the bacterial background lawn at the concentration of 5000 μg/plate in the presence of S9-mix in tester strain TA1537 (second experiment).

 

In strain TA98, fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, the reductions are not considered to be caused by toxicity of the test substance.

 

In both mutation assays, no increase in the number of revertants was observed upon treatment with Triethanol amineacetate under all conditions tested.

Table1 Experiment 1: Mutagenic response of Triethanol amineaceate in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain
	TA1535	TA1537	TA98	TA100	WP2uvrA
Without S9 -mix
positive control	735± 16	312± 40	1109± 31	1033± 50	955± 164
solvent control	10± 5	3± 1	15± 2	112± 9	22± 6
3				102± 2	26± 1
10				117± 7	32± 3
33				114± 2	21± 8
100	8± 1	3± 0	15± 4	109± 6	19± 3
333	6± 3	3± 1	17± 3	110± 4	19± 4
1000	6± 3	3± 0	12± 2	111± 12	21± 6
3330	7± 1	3± 1	11± 4	106± 10	22± 2
5000	9± 1	3± 1	14± 4	102± 4	19± 2
With S9 –mix
positive control	274± 9	187± 30	1204± 19	1421± 45	421± 21
solvent control	8± 2	3± 1	17± 4	83± 13	23± 2
3				88± 14	28± 23
10				115± 10	28± 3
33				124± 2	30± 1
100	5± 2	3± 0	14± 3	113± 4	18± 3
333	5± 3	3± 0	16± 2	114± 14	17± 4
1000	4± 1	3± 1	18± 3	113± 11	19± 3
3330	4± 1	3± 1	20± 1	95± 11	20± 2
5000	4± 2	4± 1	14± 4	105± 13	15± 5

Table 2 Experiment 2: Mutagenic response of Triethanol amineacetate in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonells typhimurium and one Escherichia coli strain
	TA1535	TA1537	TA98	TA100	WP2uvrA
Without S9 -mix
positive control	894± 39	298± 12	1105± 44	977± 45	1206± 65
solvent control	13± 3	6± 2	20± 4	107± 5	26± 4
100	9± 2	6± 2	22± 1	100± 10	21± 3
333	15± 5	6± 2	18± 5	109± 10	23± 4
1000	12± 3	5± 1	17± 4	110± 12	18± 2
3330	13± 3	5± 3	20± 3	100± 11	20± 2
5000	13± 2	5± 2	15± 1	101± 5	19± 4
With S9 –mix
positive control	143± 5	341± 94	597± 26	1153± 76	219± 46
solvent control	9± 3	4± 1	19± 3	66± 4	18± 2
100	8± 3	4± 2	25± 4	61± 2	25± 4
333	7± 1	4± 1	25± 1	65± 4	19± 1
1000	7± 4	4± 1	23± 1	65± 4	24± 1
3330	9± 4	3± 1	23± 6	63± 2	18± 3
5000	9± 1	4± 2	29± 6	59± 5	23± 6

Conclusions:

Interpretation of results (migrated information):
negative

All bacterial strains (S. typhimuriumand E.coli) showed negative responses (i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments) at up to 5 mg/plate, with and without S9, indicating that TEA acetate is not mutagenic in this assay.

Executive summary:

Evaluation of the mutagenic activity of Triethanol amineacetate in the Salmonella typhimurium reverse mutation and the Escherichia coli reverse mutation assay (with independent repeat) was conducted according to OECD guidelines (and to GLP).

 

Triethanol amineacetate was tested in the S. typhimurium assay with histidine-requiring strains of S. typhimurium (TA1535, TA1537, TA98 and TA100) and in the E. coli reverse mutation assay with tryptophan-requiring strain of E. coli (WP₂uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of Phenobarbital and β-naphthoflavone).

 

In the dose range finding test, Triethanol amineacetate was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP₂uvrA. Triethanol amineacetate did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

 

Based on the results of the dose range finding test, Triethanol amineacetate was tested in the first mutation assay at a concentration of range of 100 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. In an independent repeat of the assay with additional parameters, Triethanol amineacetate was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98 and WP₂uvrA.

Cytotoxicity, as evidenced by a decrease of the bacterial background lawn, was observed in tester strain TA1537 in the presence of S9-mix (second mutation assay) at the highest tested concentration. In all tester strains, the bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

 

Triethanol amineacetate did not induce a significant dose-related increase in the number of revertant (His*) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp⁺) colonies in tester strain WP₂uvrA both in the absence and presence of S9-metbolic activation. These results were confirmed in an independently repeated experiment.

 

In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

 

Based on the results of this study, it is concluded that Triethanol amineacetate is not mutagenic in the S. typhimurium reverse mutation assay and in the E. coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

No data are available on the genetic toxicity of DEA acetate, but a number of guideline, GLP, studies are available on structurally-related read-across compounds.

 

TEA acetate was not mutagenic in a bacterial reverse mutation assay in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP₂uvrA, at up to 5000 μg/plate, in the presence and absence of metabolic activation (S9) (Verspeek-Rip, 2010a). RA2 was not mutagenic in S. typhimurium strains TA1535, TA1537, TA98 and TA100, at up to 10,000 μg/plate, with or without S9 (Prezioso, 1996c). In mouse lymphoma (L5178Y) cells, TEA acetate was not mutagenic when tested at up to 2092 μg/ml, with or without S9 (Verspeek-Rip, 2010b).

 

No clastogenic activity (chromosome aberration) or polyploidy was seen in Chinese hamster lung (V79) fibroblast cells incubated with RA1 or RA2 at up to 5000 μg/ml, in the presence or absence of S9 (Czich, 1997, 2000).

As no positive findings were seen in any of the in vitro assays, in vivo studies were not required according to Regulation (EC) No 1907/2006.

Justification for selection of genetic toxicity endpoint
GLP, OECD guideline study (reliability 2).

Justification for classification or non-classification

According to Regulation (EC) No. 1272/2008, a substance is to be classified as a germ cell mutagen based on a weight of the evidence approach, evaluating data including in vitro tests on somatic cells. No evidence of mutagenicity was seen in read-across in vitro tests on S. typhimurium, E coli or mouse lymphoma cells (Prezioso, 1996c; Verspeek-Rip, 2010a, b). Similarly, no evidence of chromosome aberrations were seen in Chinese hamster cells in vitro (Czich, 1997, 2000). On the basis of this data, on structurally-related read-across compounds, DEA acetate does not require classification.