Di(µ-2,2´,2´´-nitrilotris(ethanol)-diperchlorato)dinatrium

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:

August 8th to 17th, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21st, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

as of May 19th, 2000

Deviations:

no

Principles of method if other than guideline:

SOP 118 008 03, edition 6, 13 November 2006, Maron D., Ames, B., 'Revised methods for the Salmonella mutagenicity test', Mut. Research 113 (1983), 173 - 215.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

480-340-8

EC Name:

-

Cas Number:

156157-97-0

Molecular formula:

C12H30Cl2N2Na2O14

IUPAC Name:

Di(µ-2,2´,2´´-nitrilotris(ethanol)-diperchlorato)dinatrium

Method

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 97a, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

other: TA97, TA98, TA 100 and TA102 contain pKM 101 and rfa; TA97, TA98 and TA100 contain urvB.

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal fraction (S9) mix

Test concentrations with justification for top dose:

50; 150; 500; 1502; 5005 μg/plate (plate incorporation test)
1250; 2499; 4998 μg/plate (pre-incubation test)

Vehicle / solvent:

- Vehicle used: deionised water.
- Justification for choice of vehicle: the test item is completely solube in water and water does not have any effects on the viability of the bacteria or the number of spontaneous revertants.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-1,2-phenylene diamine

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and water

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-amino-anthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

CULTURE OF BACTERIA: the strains were stored as stock cultures at - 80 °C. Twelve hours before the start of each experiment, one vial per strain to be used was thawed and put into a culture vessel containing 70 ml nutrient broth. After an overnight incubation (12 hours) at 37 °C, the cultures were used in the test. During the test, the cultures were stored at room temperature as to prevent changes to the titre.

S9-MIX AND S9 PREPARATION: the S-9 mix consisted of 22.5 ml phosphate buffer, 1.0 ml 0.1 m NADP-solution, 0.125 ml 1m-G6P-solution, 0.5 ml salt solution and 1.0 ml rat liver S9, 4 %. The S9 mix was freshly prepared and stored at 0 °C for each experiment. The S9 was produced from livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254 mg/kg intraperitoneally.

PREPARATION BEFORE THE TEST: in the days before each test, the media and solutions were prepared. Two days before the test, the plates were sterilised and the first batches poured. On the day before the test the remaining plates were poured. On the day of the test, the overnight cultures were checked for growth. The incubation chamber was heated to 37 °C. The water bath and the heating block were turned to 43 °C. The table surface was disinfected.

PREPARATION OF TEST SOLUTION: on the day of the start of the experiment, a stock solution containing nominally 50 g/l the test item in deionised warer was prepared. This stock solution was used to prepare the geometric series of the concentrations to be tested. Each solution was membrane filtrated to accomplish sterility.

FIRST EXPERIMENT
- Incubation time: 48 hours.
- Incubation temperature: 37 °C.
- Method of application: plate incorporation.
- Number of replicates: per strain and dose, four replicates with and four plates without S9 mix were used.
- Description of method: 10 ml of test solution of the appropriate concentration were membrane filtrated into sterile vessels. Top agar basis was melted in a microwave oven, after which, 10 ml of histidine-biotin-solution 0.5 mmol per 100 ml basis was added and the bottle was placed in the water bath at 45 °C. 0.1 ml of the appropriate solution of the test item were given into a sterile tube. After mixing with 0.1 ml overnight culture of the respective strain and 0.5 ml phosphate buffer (only for treatments without S9) or 0.5 ml S9 mix, 2 ml top-agar were added. The mixture was gently vortexed, then poured on a minimal glucose plate and distributed evenly, using a Drigalski spatula. The plates were closed, covered with brown paper and left to harden for a few minutes, then inverted and placed in the dark incubator at 37 °C. The colonies were then counted visually.

SECOND EXPERIMENT
- Incubation time: 48 hours.
- Incubation temperature: 37 °C.
- Method of application: pre-incubation.
- Number of replicates: per strain and dose, four replicates with and four plates without S9 mix were used.
- Description of method: 10 ml of test solution of the appropriate concentration were membrane filtrated into sterile vessels. Top agar basis was melted in a microwave oven, after which, 10 ml of histidine-biotin-solution 0.5 mmol per 100 ml basis was added and the bottle was placed in the water bath at 45 °C. 0.1 ml of the appropriate solution of the test item were given into a sterile tube. After mixing with 0.1 ml overnight culture of the respective strain and 0.5 ml phosphate buffer (only for treatments without S9) or 0.5 ml S9 mix were added. The mixture was incubated in an incubation chamber at 37 °C for 20 minutes. During this time the vessels were aerated through careful shaking. Then 2 ml top agar were added. The mixture was vortexed gently, then poured on a minimal glucose plate and distributed evenly, using a Drigalski spatula. The plates were closed, covered with brown paper and left to harden for a few minutes, then inverted and placed in the dark incubator at 37 °C. The colonies were then counted visually.

GENOTYPE CONFIRMATION: performed once a quarter.
- Histidine requirement: each strain was streaked on a biotin and a histidine-biotin-plate, using a sterilised wire loop.
- Ampicillin-Resistance (pKM 101) resp. ampicillin-tetracycline-resistance (pAQ1): the strains were streaked on ampicillin agar, TA102 on ampicillin-tetracylcline agar. TA1535 was taking the function of control strain since it is not ampicillin resistant.
- UV-sensitivity (urB): two plates were streaked with the five strains, and one half of the plate covered with aluminium foil so that one half of each streak was protected against light. The plates were irradiated for 8 seconds with a germicidal lamp (254 nm, 30 W), keeping a distance of 33 cm. Incubation over night at 37 °C followed.
- Crystal violet sensitivity (deep rough): for each strain two plates were used. 0.1 ml of bacteria suspension were mixed with 2 ml top-agar and poured on nutrient agar. Sterile paper discs (9 mm diameter), each soaked with 10 μl crystal violet solution (0.1 %) were placed into the middle of each plate followed by incubation over night.
- Spontaneous revertants: four repliactes, with/without S9, for each solvent which was used in the test.

DETERMINATION OF TITRE: the titre was determined by dilution of the overnight culture using sodium chloride solution and placing 0.1 ml on maximal soft agar.

TOXICITY CONTROL: performed analogously to the titre with the maximum dose of test item with and without S9 on maximal soft agar. Plate incorporation method applied, incubation time was 48 hours at 37 °C.

STERILITY CONTROL: performed analogously to the test with solvent only and S9 (without adding bacteria) on top agar.

Evaluation criteria:

A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor ≥ 2) in at least one strain is observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.

Statistics:

Mean values, standard deviations and increase factor of relevant induction were calculated.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 1535, TA 97a, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Toxicity: no signs of toxicity towards the tested strains could be observed. The background lawn was visible and the number of revertant colonies was not reduced.
- The treatements for the confirmation of the genotype, the sterility control and the determination of the titre did not show any inconsistencies.
- Mutagenicity: no significant increase of the number of revertant colonies in the treatment with and without metabolic activation could be observed. Only in strain TA1535 without metabolic activation in the first experiment an increase in the number of revertant colonies was observed. This increase was not concentration-related and barely reached the threshold of the induction factor 2.0. In the verification experiment, no increase in revertants was detected for TA1535 without metabolic activation therefore the increase observed in the first experiment was assessed as not significant. No concentration-related increase over the tested range was found.
- Histidine requirement, ampicillin-tetracyclinee-resist (pKM 101, pAQ1), UV-sensitivity (urB) and crystal violet sensitivity assessement fullfilled the criteria.

HISTORICAL CONTROL DATA
- Positive historical control data: for positive controls for all the strains the revertants were > 1000.
- Negative (solvent) historical control data: for DMSO: 94-190 (TA97a, -S9), 98-182 (TA 97a, + S9), 9-20 (TA98, - S9), 6-27 (TA98, + S9), 105-204 (TA100, -S9), 106-179 (TA100, + S9), 120-267 (TA102, -S9), 117-277 (TA102, +S9), 9-18 (TA1535, - S9), 5-21 (TA1535, + S9). For water: 119-160 (TA97a, -S9), 151-179 (TA 97a, + S9), 11-22 (TA98, - S9), 12-19 (TA98, + S9), 110-177 (TA100, -S9), 131-137 (TA100, + S9), 179-242 (TA102, -S9), 202-255 (TA102, +S9), 8-26 (TA1535, - S9), 20-21 (TA1535, + S9).

Any other information on results incl. tables

The mean revertant of the four replicates in the first experiment are presented in the table below.

Table: mean revertants first experiment.

Strain		97a		98		100		102		1535
Induction		- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
H2O	Mean	160	179	22	19	160	131	179	218	11	20
	sd	33.1	22.0	4.1	1.7	27.3	29.5	31.0	16.2	4.5	7.4
DMSO	Mean	190	158	20	18	204	179	202	181	18	21
	sd	22.8	30.6	10.6	8.5	20.4	29.8	35.0	48.1	7.0	2.6
Pos. Control	Mean	1001	1001	1001	1001	1001	1001	1001	1001	1001	1001
	sd	0	0	0	0	0	0	0	0	0	0
	f(l)	5.27	6.34	50.05	55.61	6.26	5.59	4.96	5.53	91.00	47.67
5005 μg/pl.	Mean	198	153	14	17	169	124	183	216	20	15
	sd	12	14	3	7	28	18	45	21	6	2
	f(l)	1.24	0.85	0.64	0.89	1.06	0.95	1.02	0.99	1.82	0.75
1502 μg/pl.	Mean	187	140	17	17	129	186	184	190	23	12
	sd	23	33	9	5	26	40	25	26	11	4
	f(l)	1.17	0.78	0.77	0.89	0.81	1.42	1.03	0.87	2.09	0.60
501 μg/pl.	Mean	176	148	17	20	169	181	203	161	15	15
	sd	38	21	3	6	45	23	46	61	5	4
	f(l)	1.10	0.83	0.77	1.05	1.06	1.38	1.13	0.74	1.36	0.75
150 μg/pl.	Mean	206	150	16	19	132	174	164	151	14	17
	sd	10	28	7	5	22	13	12	20	7	7
	f(l)	1.29	0.84	0.73	1.00	0.83	1.33	0.92	0.69	1.27	0.85
50 μg/pl.	Mean	195	172	16	14	135	143	192	161	22	25
	sd	50	16	7	6	32	26	71	9	2	10
	f(l)	1.22	0.96	0.73	0.74	0.84	1.09	1.07	0.74	200	1.25

The mean revertant of the four replicates in the second experiment are presented in the table below.

Table: mean revertants second experiment.

Strain		97a		98		100		102		1535
Induction		- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
H2O	Mean	119	151	11	12	155	137	180	202	26	21
	sd	31.6	53.5	3.8	2.5	46.3	10.3	30.6	17.5	8.1	8.8
DMSO	Mean	126	119	15	14	174	122	143	204	13	16
	sd	36.7	32.2	3.4	3.0	22.8	3.7	10.7	12.6	4.3	8.7
Pos. Control	Mean	1001	1001	1001	1001	1001	1001	1001	1001	1001	1001
	sd	0	0	0	0	0	0	0	0	0	0
	f(l)	7.94	8.41	66.73	71.50	6.46	8.20	7.00	4.91	38.50	62.56
4998 μg/pl.	Mean	152	131	11	10	128	115	151	184	15	15
	sd	45	45	5	2	21	15	4	26	6	8
	f(l)	1.28	0.87	1.00	0.83	0.83	0.84	0.84	0.91	0.58	0.71
2499 μg/pl.	Mean	140	105	13	8	141	133	201	154	20	13
	sd	30	33	4	1	23	37	29	28	7	3
	f(l)	1.18	0.70	1.18	0.67	0.91	0.97	1.12	0.76	0.77	0.62
1250 μg/pl.	Mean	134	114	10	9	148	125	194	154	13	18
	sd	27	42	3	2	33	14	12	47	7	1
	f(l)	1.13	0.75	0.91	0.75	0.95	0.91	1.08	0.76	0.5	0.86

1001 represents > 1000.

Applicant's summary and conclusion

Conclusions:

The substance is not mutagenic under the tested conditions with and without metabolic activation.

Executive summary:

The substance was tested for its mutagenic effects to Salmonella typhimunum strains (TA97a, TA 98, TA 100, TA 102, TA 1535) in a test by the plate incorporation and a verification test by using the pre-incubation method, according to the OECD Guideline 471 and EU Method B.13/14. The test was performed with and without the addition of rat-liver homogenate metabolising system. The compound was tested at concentrations in the range of 50 - 5005 μg/ml and 1250 - 4998 μg/ml in the first and second test respectively. Negative (plates containing no compound but only the vehicles used) and positive controls (plates containing a known mutagen) were used in parallel with the test material.

Only in strain TA1535 without metabolic activation in the first experiment an increase in the number of revertant colonies was observed. This increase was not concentration-related and barely reached the threshold of the induction factor 2.0. In the verification experiment, no increase in revertants was detected for TA1535 without metabolic activation therefore the increase observed in the first experiment was assessed as not significant. No concentration-related increase over the tested range was found. No significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains in any of the dose level tested, with and without metabolic activation.

Conclusion

The substance was found to be non-mutagenic under the conditions of this test.