Bis(2-hydroxyethyl)ammonium acetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

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:

2010-02-23 to 2010-04-13

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted according to Guidelines in a GLP certified laboratory, on a structurally closely-related substance

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

1. In the dose range-finding study, the test substance was tested at a concentration of above 0.01 M. 2. When culturing the cells, the humidity and temperature were occasionally outside the recommended range

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

See deviations above

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: The recommendations of the "International Workshop on Genotoxicity Tests Workgroup", published in the literature (Clive et. al, 1995, Moore et al, 1999, 2000, 2002, 2003, 2006 and 2007)

Deviations:

yes

Remarks:

See deviations above

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Triethanolamine acetate

IUPAC Name:

Triethanolamine acetate

Constituent 2

Reference substance name:

Tris(2-hydroxyethyl)ammonium acetate

EC Number:

238-874-2

EC Name:

Tris(2-hydroxyethyl)ammonium acetate

Cas Number:

14806-72-5

IUPAC Name:

2-hydroxy-N,N-bis(2-hydroxyethyl)ethanaminium acetate

Test material form:

other: solid

Details on test material:

Identification: Triethanol amineacetate
Molecular formula: C8H19NO5
Molecular weight: 209.24
CAS Number: 14806-72-5
Smiles notation N(CCO)(CCO)CCO.C(C)(=O)O
InChl UPCXAARSWVHVLY-UHFFFAOYSA-N
Structural formula attached as image file: see Fig. 1
Test substance storage: At room temperature protected from light under nitrogen
Stability under storage conditions: Stable

Method

Target gene:

Thymidine kinase gene

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y/TK+/- 3.7.2C mouse lymphoma cells
Sourced from the American Type Culture Collection.
Stocks stored in liquid nitrogen until use.
Density was preferably kept below 1 x 10^6 cells/ml

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix: rat liver microsomal enzymes were routinely prepared from adult Wistar rats

Test concentrations with justification for top dose:

Dose range-finding concentrations: 0 (control), 33, 100, 333, 1000 and 2092 µg/ml
First and second mutation assay concentrations: 0 (control), 1, 3, 10, 33, 100, 333, 1000 and 2092 µg/ml

Vehicle / solvent:

RPMI 1640 medium

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: MMS was used as the positive control in cultures without metabolic activation. Cyclophosphamide (CPA) was used as the positive control in cultures with metabolic activation

Details on test system and experimental conditions:

TEST SYSTEM: L5178Y/TK+/- 3.7.2C mouse lymphoma cells

METHOD OF APPLICATION: The test substance was dissolved in RPMI 1640 medium and filter sterilised. Triethanol amineacetate concentrations were added to the mouse lymphoma cells within 30 minutes of preparation.

DURATION

- Preincubation period: Prior to addition of the test substance, mouse lymphoma cells were grown for 1 day in HAT medium to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days in RPMI 1640 medium containing hypoxanthine and thymidine only. Cells were then cultered in RPMI 1640 medium for at least 1 day before starting the experiment.

- Exposure duration: In experiment 1, cells were exposed to Triethanol amineacetate for 3 hours in the presence and absence of metabolic activation. In experiment 2, cells were exposed to Triethanol amineacetate for 3 hours in the presence of metabolic activation and 24 hours in the absence of metabolic activation.

- Expression time (cells in growth medium): Two days

- Selection time (if incubation with a selection agent): Eleven or twelve days in TFT selective medium

- Fixation time (start of exposure up to fixation or harvest of cells): Fourteen or fifteen days from start of treatment with test substance until analysis of cells

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: The experiment was performed in duplicate using eight concentrations of test substance

NUMBER OF CELLS EVALUATED: For determination of the mutation frequency, a total number of 9.6 x 10^5 cells/concentration were plated in five 96-well microtiter plates, with each well containing 2000 cells in TFT medium. At the end of the selection period, the plates were stained for 2 hours by adding 0.5 mg/ml MTT to each well. The plates were then scored with the naked eye or using a microscope for total cell count in each well.

DETERMINATION OF CYTOTOXICITY
- Method: The cytotoxicity of the test substance was measured by calculating the relative suspension growth of the cells treated with Triethanol amineacetate versus medium-treated control cultures.

Evaluation criteria:

A test substance is considered positive in the mutation assay if it induces a mutation frequency (MF) of more than MF(controls) + 126 in a dose-dependent manner. An increase should be biologically relevant in comparison with the historical control range.

A test substance is considered equivocal in the mutation assay if no clear conclusion for positive or negative results can be made after an additional confirmation study.

A test substance is considered negative in the mutation assay if:
a) None of the tested concentrations reach a mutation frequency of MF(controls) + 126 AND
b) The results are confirmed in an independently repeated test.

Statistics:

No statistical analysis was required.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The spontaneous mutation frequencies in the vehicle-treated control cultures were between the minimum and maximum values of the historical control range

The results of the gene mutation assay were confirmed through an independent confirmatory assay.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary cytotoxicity test

 

The initial dose range-finding test conducted on Triethanol amineacetate indicated that the test substance was not toxic up to and including the highest test substance concentration of 2902 µg/ml, either with or without metabolic activation. In the presence of S9-mix, the relative suspension growth was 91 % at the test substance concentration of 2902 µg/ml, and in the absence of S9-mix, the relative suspension growth was 55 % at 2902 µg/ml compared to that of the solvent control.

 

Triethanol amineacetate did not precipitate in the exposure medium at any of the concentrations tested.

 

Mutagenic assays

 

In experiments I and II, no toxicity was observed at any dose level, either in the presence or absence of metabolic activation.

 

In experiment I, the concentration of S9-mix in the exposure medium was 8 % (v/v). No significant increase in the number of mutant colonies was observed after treatment with Triethanol amineacetate in either the presence or absence of S9-mix.

 

In experiment II, the concentration of S9-mix in the exposure medium was increased to 12 % (v/v). The cells were treated with the test substance for 3 hours in the presence of S9-mix, or 24 hours in the absence of S9-mix. No significant increase in the number of mutant colonies was observed after treatment with Triethanol amineacetate in either the presence or absence of S9-mix.

 

MMS and CPA were used as positive controls for cells treated in the absence and presence of S9-mix respectively. These substances significantly increased the mutation frequency at the TK locus, indicating that the assay was appropriate for the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned properly. Furthermore, the observed mutation frequencies of the positive control substances were within the acceptability criteria of this assay.

Table 1: Cytotoxicity data from dose-range finding test

 

Concentration (µg/ml)	S9 Mix	Treatment period (h)	Number of cells at 3 h (cells/ml x 10^6)	Number of cells/ml at 24 h (cells/ml x 10^6)	Number of cells/ml at 48 h (cells/ml x 10^6)	SG (x 10^5cells/ml)	RSG (%)
0*	-	3	5.2	7.0	6.1	141	100
33	-	3	5.5	6.9	5.7	139	99
100	-	3	5.2	7.0	5.9	136	97
333	-	3	5.2	6.6	6.0	131	9.
1000	-	3	5.3	6.7	5.4	124	88
2902	-	3	4.7	7.9	5.2	122	87
0*	+	3	4.8	6.7	6.8	141	100
33	+	3	4.8	7.0	6.0	128	91
100	+	3	4.9	6.1	6.4	122	87
333	+	3	4.4	7.6	6.3	134	95
1000	+	3	4.2	7.8	6.0	126	89
2902	+	3	5.1	6.8	5.8	128	91
0*	-	24	-	6.8	4.7	25	100
33	-	24	-	7.4	4.2	25	97
100	-	24	-	7.4	4.0	24	93
333	-	24	-	7.8	3.6	23	89
1000	-	24	-	7.0	3.6	20	79
2902	-	24	-	5.8	3.0	14	55

* = solvent control

SG = suspension growth

RSG = relative suspension growth

Table 2: Cytotoxic and mutagenic response of Triethanol amineacetate in experiment I

 

Dose     (µg/ml)	Relative suspension growth (%)	Cloning efficiency   (day 2)	Relative Survival   (day 2)	Relative total growth   (%)	Mutation frequency per 10^6cells
					Total	Small	Large
Without metabolic activation 3 hours treatment
SC1	100	77	100	100	72	38	32
SC2	100	74	100	100	58	26	30
1	103	67	89	92	77	24	51
3	106	62	83	87	101	50	48
10	103	52	69	71	92	56	35
33	95	70	94	88	72	49	21
100	119	56	74	88	101	48	50
333	114	58	76	87	80	22	56
1000	117	67	89	104	60	30	28
2092	111	58	78	87	69	31	36
MMS	30	49	65	20	1064	474	453
With metabolic activation 3 hours treatment
SC1	100	102	100	100	63	27	34
SC2	100	105	100	100	52	29	22
1	96	104	100	96	59	34	23
3	121	93	89	108	90	46	41
10	91	102	99	90	47	25	21
33	112	74	71	79	131	75	50
100	102	102	99	100	70	39	28
333	89	107	103	92	58	16	41
1000	97	108	104	101	68	35	31
2092	105	83	84	84	119	49	65
CP	42	44	18	18	1487	623	626

Table 3: Cytotoxic and mutagenic response of Triethanol amineacetate in experiment II

 

Dose     (µg/ml)	Relative suspension growth (%)	Cloning efficiency   (day 2)	Relative Survival   (day 2)	Relative total growth   (%)	Mutation frequency per 10^6cells
					Total	Small	Large
Without metabolic activation 3 hours treatment
SC1	100	89	100	100	70	45	23
SC2	100	90	100	100	65	32	31
1	94	91	102	96	96	61	30
3	107	85	95	101	67	31	34
10	102	94	105	107	80	40	37
33	108	78	87	94	66	33	31
100	106	80	90	95	83	47	33
333	104	88	98	102	78	55	21
1000	81	99	111	90	56	27	28
2092	69	81	91	63	95	51	41
MMS	74	64	72	53	803	519	199
With metabolic activation 3 hours treatment
SC1	100	93	100	100	66	37	26
SC2	100	108	100	100	77	52	23
1	102	111	111	113	54	30	22
3	95	91	91	86	86	46	37
10	83	107	106	88	75	49	23
33	102	115	114	116	76	56	18
100	98	85	85	83	119	73	41
333	97	120	119	116	71	41	27
1000	106	111	111	118	65	43	20
2092	100	104	103	104	83	50	30
CP	74	88	87	64	878	474	250

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

No significant increase in the mutation frequency at the TK locus was observed in mouse lymphoma L5278Y cells after treatment with Triethanol amineacetateat non-toxic concentraitons of up to 2092 ug/ml, with and with without S9.

Executive summary:

The objective of this guideline (GLP) study was to assess the ability of Triethanol amineacetate to induce mutations at the TK locus on chromosome 11 of mouse lymphoma L5178Y cells, in both the presence and absence of a metabolic activation system (S9). A preliminary test was performed to determine the cytotoxicity of the test substance. L5278Y cells were exposed to the test item at concentrations of between 33 and 2092 µg/ml for 3 hours with and 3 and 24 hours without S9. At the end of this treatment period the cloning efficiency and relative suspension growth were determined.

 

To assess the capacity of Triethanol amineacetate to induce mutations at the TK locus, a mutation assay was performed in two independent experiments. In both experiments, L5178Y cells were treated with 1, 3, 10, 33, 100, 333, 1000 and 2092 µg/ml. In experiment I, cells were exposed to the test item for 3 hours with and 3 and 24 hours without metabolic activation using 8 % v/v S9-mix. In experiment II, cells were exposed to the test item for 3 hours with and 3 and 24 hours without metabolic activation using 12 % v/v S9-mix. Cultures treated with medium only were used as solvent controls. MMS was used as a positive control substance in cultures without metabolic activation, while CPA was used as a positive control substance in cultures with metabolic activation using S9 mix. The TFT-resistant mutant frequency per 10⁶ cloneable cells was used to determine the potential mutagenic effects of the test substance.

 

Treatment of mouse lymphoma L5278Y cells with Triethanol amineacetate did not induce toxicity as measured by relative suspension growth. No significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance at any of the concentrations tested. The negative and positive control values were within 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 at the TK locus of mouse lymphoma L5178Y cells.