N,N,N',N'-tetramethyl-2,2'-oxybis(ethylamine)

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:

1982-07-21 - 1982-07-23

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The study report included thorough methods and results sections, followed the internal laboratory protocol without any reported deviations, and generally followed the related OECD Method (OECD 476). The study was said to be conducted under GLP conditions but no certificate was provided.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1982

Report date:

1982

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Study did not include cross-linking mutagens (TA102) or a DNA repair-proficient strain of E. coli.

Principles of method if other than guideline:

An internal BRCC Laboratory protocol was followed with no deviations.

GLP compliance:

yes

Remarks:

As described in Section I. Quality Control (no certificate)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N,N',N'-tetramethyl-2,2'-oxybis(ethylamine)

EC Number:

221-220-5

EC Name:

N,N,N',N'-tetramethyl-2,2'-oxybis(ethylamine)

Cas Number:

3033-62-3

Molecular formula:

C8H20N2O

IUPAC Name:

{2-[2-(dimethylamino)ethoxy]ethyl}dimethylamine

Test material form:

liquid

Specific details on test material used for the study:

- Name of test material (as cited in study report): NIAX Catalyst A-99
- Substance type: active substance
- Physical state: Liquid
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: not applicable
- Isomers composition: not applicable
- Purity test date: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: Room temperature
- Other: Storage location was chemical hood/Genetic Toxicology Department
- Lot/batch No.: 51-DKE-115

Method

Target gene:

Reverse mutations in the histidine locus

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):

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S-9 fraction of rat liver homogenate obtained from Arcolor 1254-treated Sprague Dawley male rats

Test concentrations with justification for top dose:

0.003, 0.01, 0.03, 0.1, 0.3 µL/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water, deionized, sterile
- Justification for choice of solvent/vehicle: no data

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

without activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without activation

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without activation

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation)

DURATION
- Preincubation period: Broth cultures are prepared by inoculating from the master plates into nutrient broth and incubated overnight with agitation.
- Exposure duration: 48-72 hours

NUMBER OF REPLICATIONS: Each dose tested in triplicate and with all 5 bacterial strains

DETERMINATION OF CYTOTOXICITY
- Method: Inhibition of growth of background lawn

OTHER EXAMINATIONS: not applicable

OTHER: A preliminary toxicity test was performed using strain TA100 to determine the level of toxicity of the test substance. Ten doses were tested for toxicity with a plate assay performed in the manner used for mutagenicity determinations. Toxicity was assessed at 24 to 48 hours after treatment by either growth inhibition of the background lawn or a reduction in the number of spontaneous mutants.

Evaluation criteria:

The spontaneous reversion for the solvent controls must be within the laboratory's historical range. The positive controls must demonstrate that the test systems are responsive with known mutagens. A test chemical is considered a bacterial mutagen if the number of revertant colonies is at least twice the solvent control for at least one dose level and there is evidence of a dose-related increase in the number of revertant colonies. If a test chemical produces a marginal or weak response that cannot be reproduced, the initial positive result loses significance. If there is no evidence of a dose-related increase in the number of revertant colonies and the number of revertant colonies is not twice the solvent control, then the test chemical is not considered to be a bacterial mutagen.

Statistics:

All plate counts and the respective means and standard deviations were provided. If the background lawn is sparse and the colony count is still recorded, that number is not used in the calculation of the mean number of colonies and its standard deviation.

Results and discussion

Test results

Test results 1

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:

cytotoxicity

Remarks:

Some toxicity was noted at the 0.3 uL dose.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Some toxicity was noted at the 0.3 uL dose.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not applicable
- Effects of osmolality: not applicable
- Evaporation from medium: not applicable
- Water solubility: not applicable
- Precipitation: not applicable
- Other confounding effects: Concurrently run sterility checks showed that the S9 mix, PBS, the test chemical and all solvents and controls were sterile.

RANGE-FINDING/SCREENING STUDIES: The top dose of the 50 uL/plate inhibited all growth, while the doses of 10, 3, 1, and 0.3 microliters per plate allowed only sparse growth.

COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous reversion rate of the negative controls was within the historical range at the laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxicity, observed as a reduction of the background lawn, was obtained with at least one plate for each strain at the 0.3 uL per plate dose in both tests. Those plate counts were not used to calculate the means.

See Tables 1 and 2 below for full results.

Any other information on results incl. tables

Table 1. Results of the Salmonella Mutagenicity Assay. NIAX Catalyst A-99, Without Activation

Test Agent	Dose per Plate	Plate Counts			Average	Std. Dev.
		1	2	3
Strain TA98
Solvent	100 µL	20	15	16	17	2.6
POS: 01	10 µg	810	869	840	840	29.5
Test Substance	0.3 µL	15	21	16 (T)	18	4.2
	0.1 µL	15	18	14	16	2.1
	0.03 µL	20	21	14	16	3.8
	0.01 µL	15	7	13	12	4.2
	0.003 µL	12	15	7	11	4.0
Strain TA100
Solvent	100 µL	161	148	143	151	9.3
POS: 02	10 µg	1338	1172	869	1126	237.8
Test Substance	0.3 µL	155	140 (T)	149 (T)	155	----
	0.1 µL	136	158	141	145	11.5
	0.03 µL	115	156	143	138	21.0
	0.01 µL	123	160	144	142	18.6
	0.003 µL	132	158	145	145	13.0
Strain TA1535
Solvent	100 µL	51	37	36	41	8.4
POS: 02	10 µg	1341	1344	1371	1352	16.5
Test Substance	0.3 µL	43	42	28 (T)	42	0.7
	0.1 µL	21	28	55	35	18.0
	0.03 µL	45	42	41	43	2.1
	0.01 µL	42	37	43	41	3.2
	0.003 µL	34	50	24	36	13.1
Strain TA1537
Solvent	100 µL	4	7	6	6	1.5
POS: 03	60 µg	332	350	328	337	11.7
Test Substance	0.3 µL	6	9	3 (T)	8	2.1
	0.1 µL	2	7	4	4	2.5
	0.03 µL	3	4	10	6	3.8
	0.01 µL	5	4	6	5	1.0
	0.003 µL	4	4	7	5	1.7
Strain TA1538
Solvent	100 µL	6	5	5	5	0.6
POS: 01	10 µg	1155	1229	1249	1211	49.5
Test Substance	0.3 µL	6	3 (T)	2 (T)	6	----
	0.1 µL	12	8	12	11	2.3
	0.03 µL	5	8	7	7	1.5
	0.01 µL	5	7	4	5	1.5
	0.003 µL	5	4	3	4	1.0

T: Toxic

POS: Positive Controls: 01: 4-nitro-o-phenylenediamine; 02: Sodium azide; 03: 9-aminoacridine; 04: 2-aminoanthracene

Table 2. Results of the Salmonella Mutagenicity Assay, NIAX Catalyst A-99, With Activation

Test Agent		Plate Counts			Average	Std. Dev.
	Dose per Plate	1	2	3
Strain TA98
Solvent	100 µL	39	32	33	35	3.8
POS: 04	10 µg	1793	1549	1431	1591	184.6
Test Substance	0.3 µL	32	33	28 (T)	32	0.7
	0.1 µL	24	32	22	26	5.3
	0.03 µL	28	33	28	30	2.9
	0.01 µL	25	28	13	22	7.9
	0.003 µL	25	21	31	26	5.0
Strain TA100
Solvent	100 µL	97	101	108	102	5.6
POS: 04	10 µg	1429	1226	1299	1318	102.8
Test Agent	0.3 µL	93 (T)	90 (T)	96	96	---
	0.1 µL	102	110	125	112	11.7
	0.03 µL	113	89	90	97	13.6
	0.01 µL	117	110	89	105	14.6
	0.003 µL	112	110	97	106	8.1
Strain TA1535
Solvent	100 µL	15	9	14	13	3.2
POS: 04	10 µg	99	88	98	95	6.1
Test Substance	0.3 µL	8 (T)	15	12 (T)	15	---
	0.1 µL	16	9	14	13	3.6
	0.03 µL	4	17	8	10	6.7
	0.01 µL	12	7	9	9	2.5
	0.003 µL	5	11	7	8	3.1
Strain TA1537
Solvent	100 µL	3	9	6	6	3.0
POS: 04	10 µg	139	117	181	146	32.5
Test Substance	0.3 µL	2 (T)	3 (T)	2 (T)	---	---
	0.1 µL	9	4	9	7	2.9
	0.03 µL	8	5	10	8	2.5
	0.01 µL	2	9	6	6	3.5
	0.003 µL	10	4	8	7	3.1
Strain TA1538
Solvent	100 µL	14	29	17	20	7.9
POS: 04	10 µg	543	789	294	542	247.5
Test Substance	0.3 µL	10	26	7 (T)	18	11.3
	0.1 µL	9	15	15	13	3.5
	0.03 µL	17	13	26	19	6.7
	0.01 µL	16	24	8	16	8.0
	0.003 µL	18	12	17	16	3.2

T: Toxic

POS: Positive Controls - 01: 4-nitro-o-phenylenediamine; 02: Sodium azide; 03: 9-aminoacridine; 04: 2-aminoanthracene

Applicant's summary and conclusion

Conclusions:

NIAX Catalyst A-99 did not produce a dose-dependent mutagenic response in any of the Salmonella typhimurium strains. Under the conditions of this assay, NIAX Catalyst A-99 was not mutagenic at 0.003, 0.01, 0.03, 0.1, and 0.3 uL/plate in the Salmonella/microsome mutagenicity assay.