Trisodium 2-(carboxylatomethyl(2-hydroxyethyl)amino)ethyliminodi(acetate)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990-01-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium 2-(carboxylatomethyl(2-hydroxyethyl)amino)ethyliminodi(acetate)

EC Number:

205-381-9

EC Name:

Trisodium 2-(carboxylatomethyl(2-hydroxyethyl)amino)ethyliminodi(acetate)

Cas Number:

139-89-9

Molecular formula:

C10H18N2O7.3Na

IUPAC Name:

trisodium [{2-[bis(carboxylatomethyl)amino]ethyl}(2-hydroxyethyl)amino]acetate

Details on test material:

- Name of test material (as cited in study report): Trilon D flussig
- Lot/batch No.: B110
- Storage condition of test material: 4 - 8 degrees C
- All other template details: Not reported

Method

Target gene:

his+ revertants

Species / strain

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 checked for Mycoplasma contamination: no
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
- Periodically checked for deep rough character: yes
- Periodically checked for UV sensitivity: yes
- Periodically checked for ampicillin resistance (R factor plasmid): yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

0, 20, 100, 500, 2500, 500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: aqua dest.
- Justification for choice of solvent/vehicle: complete solubility of the test substance in aqua dest.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: with S-9 mix: 2-aminoanthracine (in DMSO); without S-9 mix: N-methyl-N'-nitro-N-nitrosoguianidine (in DMSO) for strains TA 100 and TA 1535; 4-nitro-o-phenylendiamine (in DMSO) for strain TA 98; 9-aminoacridine chloride monohydrate (in DMSO) for TA 1537

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation), test tubes containing 2 mL portions of soft agar (100 ml agar - 0.6% agar + 0.6% NaCl) and 10 mL amino acid solution are kept in a water bath at 45 degrees C. The remaining ingredients were added: 0.1 mL test solution, 0.1 mL bacterial suspension, 0.5 mL S-9 mix (in tests with metabolic activation) or 0.5 ml phosphate buffer (in tests without metabolic activation)

DURATION
- Preincubation period: 20 minutes for that study
- Exposure duration: 48 hours at 37 degrees C

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: >10^7 cells

DETERMINATION OF CYTOTOXICITY
- Method: reduced his- background growth

OTHER EXAMINATIONS: No

Other: Tissue preparation for the S-9 fraction (according to Ames et al.)
- 5 male Sprague-Dawley rats (200-300 g) receive a single IP injection of 500 mg Aroclor 1254 per kg BW 5 days before sacrifice
- Rats are sacrificed, livers removed, weighted and washed in a 150 mM KCl solution
- Livers then are cut into small pieces and homogenized in three volumes of KCl solution and centrifuged at 9000 x g for 10 minutes at 4 degrees C
- 5 ml portions of the supernatant are quickly deep-frozen in dry ice and stored at -70 to -80 degrees C for up to 2 months
- S9 mix is prepared freshly prior to each experiment with: MgCl2 - 8 mM, KCl - 33 mM, glucose-S-phosphate - 5 mM, NADP - 4 mM, phosphate buffer (pH 7.4) - 100 mM

Evaluation criteria:

1) doubling of the spontaneous mutation rate (control), 2) dose-response relationship, 3) reproducibility of the results

Statistics:

Not reported

Results and discussion

Test results

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:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Full results provided on Tables 1-4 showing the number of revertants/plate for each strain.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Results for Strain TA1535 – Number of Revertants/Plate.

Dose	without S9	Ave.	SD	with S9	Ave.	SD
Negative control Aqua Dest.	12	16	4	14	13	1
	18			12
	19			13
20	16	15	1	13	15	3
	14			18
	14			13
100	15	18	3	13	15	4
	20			19
	18			12
500	19	16	3	10	14	5
	15			20
	14			12
2500	15	16	2	14	12	4
	19			15
	15			8
5000	13	14	1	12	14	3
	15			17
	15			13
Pos. Control 2-AA				130	114	17
				96
				115
Pos. Control MNNG	1880	1927	64
	1900
	2000

 

Table 2. Results for Strain TA100 – Number of Revertants/Plate.

Dose	without S9	Ave.	SD	with S9	Ave.	SD
Negative control Aqua Dest.	100	105	6	119	112	13
	102			97
	112			121
20	124	131	13	137	126	10
	123			123
	146			117
100	134	119	14	127	123	9
	117			129
	106			113
500	85	85	1	108	111	7
	84			119
	86			106
2500	100	114	12	111	115	4
	124			116
	117			118
5000	124	144	19	133	127	7
	146			130
	162			119
Pos. Control 2-AA				1880	1913	42
				1900
				1960
Pos. Control MNNG	1900	2083	161
	2150
	2200

 

Table 3. Results for Strain TA1537 – Number of Revertants/Plate.

Dose	without S9	Ave.	SD	with S9	Ave.	SD
Negative control Aqua Dest.	5	8	3	10	12	2
	10			12
	9			13
20	12	10	2	22	18	4
	10			15
	9			18
100	11	11	1	20	15	6
	12			9
	10			15
500	12	11	4	9	11	2
	14			11
	6			13
2500	10	12	6	3	7	5
	8			12
	19			5
5000	7	10	4	8	8	1
	14			7
	10			8
Pos. Control 2-AA				204	185	19
				184
				167
Pos. Control MNNG	558	527	34
	532
	491

 

Table 4. Results for Strain TA98 – Number of Revertants/Plate.

Dose	without S9	Ave.	SD	with S9	Ave.	SD
Negative control Aqua Dest.	29	30	3	44	44	1
	28			43
	33			45
20	34	31	3	44	44	6
	31			39
	29			50
100	29	31	4	45	39	5
	36			36
	29			37
500	29	31	3	51	42	8
	35			36
	29			38
2500	42	31	12	41	48	6
	18			52
	34			52
5000	28	35	8	45	40	10
	34			29
	44			47
Pos. Control 2-AA				1460	1447	160
				1280
				1600
Pos. Control MNNG	800	815	19
	836
	810

 

Applicant's summary and conclusion

Conclusions:

There was no increase in the number of mutations (his+ revertants) in bacterial strains TA 1535, TA 100, TA 1537, and TA 98 exposed to 20-5000 ug/plate of the test material in agar for 48 hours. The test material did not appear to be cytotoxic. There was an appropriate response from the positive control materials.

Executive summary:

An increase in the number of his+ revertants was not observed both in the standard plate test and in the pre-incubation test either without S-9 mix or after the addition of a metabolizing system to strains TA 1535, TA 100, TA 1537, and TA 98 from 20 -5000 ug/plate of Trilon D. According to the results of the present study, the test substance Trilon D flussig is not mutagenic in the Ames test under the experimental conditions chosen.