2,2'-oxydi(ethylamine)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two reliable in vitro genetic toxicity studies are available: an Ames test with BAEE and a mammalian cell gene mutation assay with the read-across substance BDMAEE. All tests were negative. No in vitro cytogenicity study in mammalian cells is necessary as adequate data from an in vivo cytogenicity test with the read-across substance BDMAEE is available (REACH Annex VIII, Section 8.4.2 column 2).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-12-14 to 2012-01-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well documented GLP study performed according to OECD Guideline 471 and ICH of Technical Requirements for Registration of Pharmaceuticals for Human Use. Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Genotoxicity: A standard Battery for Genotoxicity Testing of Pharmaceuticals.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): BAEE
- Substance type: Clear, slightly yellow liquid
- Physical state: Liquid
- Analytical purity: 100%
- Lot/batch No.: 0F802
- Expiration date of the lot/batch: no data
- Storage condition of test material: Room temperature, stored protected from light

Target gene:

Histidine and tryptophan locus

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Experiment 1 (initial toxicity - mutation assay): 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg/plate (all strains tested)
Experiment 2: retest of tester strain WP2 uvrA in presence of S9 activation due to unacceptable vehicle control value in experiment 1: 50, 150, 500, 1500 and 5000 µg/plate
Experiment 3 (confirmatory mutagenicity assay): 50, 150, 500, 1500 and 5000 µg/plate
Experiment 4: retest of tester strain TA1537 in the presence of S9 due to numerous intermediate colonies that interfered with evaluation of the assay plates: 50, 150, 500, 1500 and 5000 µg per plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Water was selected as the solvent of choice based on the solubility of the test article and compatibility with the target cells. The test article formed a clear solution in water at approximately 50 mg/mL, the maximum concentration tested in the solubility test.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (1 µg/plate for TA98, TA1535, and TA1537; 2 µg for TA100, 15 µg for WP2 uvrA

Remarks:

with metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation: 1 µg for TA98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation: 1 µg for TA100 and TA1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation: 75 µg for TA1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation: 1000 µg for WP2uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 hours
- The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate.
- Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity.

NUMBER OF REPLICATIONS: All dose levels of test article, vehicle control and positive controls were plated in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: A dose level is considered toxic if one or both of the following criteria are met: (1) A > 50% reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) At least a moderate reduction in the background lawn.

Evaluation criteria:

For the test article to be evaluated positive, it must cause a dose-dependent increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article.
Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value.
An equivocal response is a biologically relevant increase in revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative, if it was neither positive nor equivocal.

Statistics:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.

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

Remarks:

Neither precipitate nor toxicity was observed

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

Remarks:

Neither precipitate nor toxicity was observed

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Water was selected as the solvent of choice based on the solubility of the test article and compatibility with the target cells. The test article formed a clear solution in water at approximately 50 mg/mL, the maximum concentration tested in the solubility test.
- Precipitation: No precipitation was observed

RANGE-FINDING/SCREENING STUDIES: The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate. No positive mutagenic responses were observed with any of the tester strains in the absence of S9 activation and with any of the Salmonella tester strains in the presence of S9 activation. Neither precipitate nor toxicity was observed. Due to an unacceptable vehicle control value, tester strain WP2uvrA in the presence of S9 activation was not evaluated for mutagenicity but was retested. No positive mutagenic response was observed with tester strain WP2 uvrA in the presence of S9 activation during retesting. Neither precipitate nor toxicity was observed.

COMPARISON WITH HISTORICAL CONTROL DATA: The number of revertants per plate for the negative and positive control substances were in the range of the historical control values of the laboratory.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Under the conditions of this study, the test article BAEE was concluded to be negative in the Bacterial Reverse Mutation Assay and is thus not mutagenic. Based on these results and according to the criteria laid down in the CLP Regulation the test substance is not to be classified as mutagenic.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1982-10-19 to 1982-11-29

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 not conducted under GLP conditions.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

not specified

Principles of method if other than guideline:

An internal BRCC Laboratory protocol was followed with no deviations.

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

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
- Lot/batch No.: 51-DKE-115

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HPRT)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Antibiotic-free Ham's Modified F12 Medium supplemented with 10% (v/v) heat-inactivated fetal bovine sera (F12-10) and lacking in hypoxanthine.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 homogenate from Aroclor-1254 induced Sprague-Dawley rats (Meloy Laboratories, Springfield, VA)

Test concentrations with justification for top dose:

Initial Plating Efficiency & Mutant Induction Test without S9 Activation: 0.08, 0.09, 0.1, 0.15, 0.2 and 0.25% v/v
Initial Plating Efficiency & Mutant Induction Test with S9 Activation: 0.08, 0.09, 0.1 and 0.15% v/v
Repeat Plating Efficiency & Mutant Induction Test with S9 Activation: 0.16, 0.17, 0.18% v/v

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Sterile, deionized water (GIBCO)
- Justification for choice of solvent/vehicle:

Untreated negative controls:

yes

Remarks:

Medium control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Dimethylnitrosamine (with activation); Ethyl methanesulfonate (without activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium. For treatment of cells without metabolic activation, F12 medium with 50 units/mL of penicillin, 50 ug/mL streptomycin, and 5% (v/v) of dialyzed bovine serum (F12-D5) is used. For treatments with metabolic activation, identical medium, but without serum, is employed.

DURATION
- Preincubation period: 20-24 hours
- Exposure duration: 5 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 6-8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15-18 days

Further details on treatment with test chemicals: Cells are inoculated 20 to 24 hours prior to treatment into flasks containing F12-D5 medium and incubated at 37 deg C in a 5 - 6% CO2 atmosphere. Appropriate concentrations of the test agent or control chemicals are added to the cells and cultures are treated for 5 hr at 37 deg C. The medium and test agents are removed by suction, cells are rinsed once or twice and fresh F12-D5 medium is added. The cells are allowed a period of 20 to 24 hours of recovery from treatment before cytotoxicity of the chemical treatment is determined. Treatment of cells with metabolic activation is performed identically, with the exception that F12 medium without serum and containing 1.0 mL of S9 activation mixture per 4.0 mL of medium is employed.

Further details on determination of mutant induction: At 2 to 3 day intervals after treatment with the various test agents, approximately 5 x 10^5 cells are subcultured in tissue culture dishes in F12-D5 medium and incubated at 37 deg C in a 5 - 6% CO2 atmosphere. After a period of at least 7 days to allow "expression" of the mutant phenotype, cells are dissociated with 0.05% to 0.075% trypsin, counted and plated at a concentration of 2 x 10^5/dish in five culture dishes (1 x 10^6 total cells) which each contain 10 mL of F12-D5 (TG) selective medium. Cells are diluted and 100 cells/dish are added to four culture plates containing F12-D5 medium (without TG) to assess viability (plating efficiency) of the treated cell population; the value obtained for the viable fraction with cells exposed to various doses and to control agents is used to correct the mutant frequency for cell populations with different growth ability. All cultures are then incubated for an additional 6 to 8 days to allow growth of cells; medium is then discarded and colonies are fixed and stained for counting. The number of colonies in selection plates and in the viability test are counted by electronic methods, checked by manual counts and data are recorded both as mutants/10^6 total cells and mutants/10^6 viable cells.

SELECTION AGENT (mutation assays): 6-thioguanine (TG) - F12-D5 medium containing 2.0 ug/mL TG is used as a "selective medium."

NUMBER OF CELLS EVALUATED: 10^6 viable cells & 10^6 total cells

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The criteria for interpretation of the test results as a positive or negative response depend upon both the level of statistical significance from the concurrent control and the evidence of a dose-response effect following treatment. Historical negative control data indicate that an average spontaneous mutation frequency in CHO cells of approximately 2 mutants/10^6 viable cells with a 95 percentile range of 0 to 18 mutants/10^6 viable cells. Tests are usually repeated to clarify the biological significance of the data when questionable results are obtained.

Statistics:

Analysis of mutation frequencies in the CHO test follow the procedure of Irr and Snee, which employs the Box-Cox Transformation to transform data prior to parametric analysis using the Student's t-test.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no data
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: It was observed that NIAX Catalyst A-99 had similar toxicity when tested with or without S9 metabolic activation and similar doses were used for each test. Data obtained on cytotoxicity from a related Sister Chromatid Exchange (SCE) test were used to select doses for the current CHO mutation test because cytotoxicity was so similar both with and without S9 activation. Concentrations of 0.2% and 0.3% (or above) produced a marked degree of lysis of CHO cells tested with and without S9, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA: The mutation frequency obtained with the medium negative control in the repeat test was outside the 95 percentile historical range for negative control values, but similar random variations with single cultures are not uncommon with CHO cells.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Table 1 presents the cytotoxicity data determined at 24 hours post-exposure for CHO cells treated with the test substance in the presence and absence of a liver S9 metabolic activation system. The test substance produced dose-related cytotoxicity to CHO cells treated without S9 metabolic activation. A steeper dose effect response was observed in the test with S9 activation over the narrow concentration range between 0.15% and 0.2%. Because only four doses allowed sufficient survival for determination of mutant induction, with S9 activation, a repeat test was performed using concentrations of 0.16% to 0.18% within the steep cytotoxic response range observed in the first test. Table 3 shows that little cytotoxicity was obtained for doses in this range and the results suggest that 0.20% is the threshold lethal dose in this steep dose-effect portion of the response curve in tests with S9 activation.

MUTATION: Table 2 presents the data for induction of mutants by the test chemical and control agents. NIAX Catalyst A-99 did not produce a dose-related increase in the frequency of mutants/10^6 viable cells over the range of concentrations tested either with or without the presence of an S9 metabolic activation system. No concentration of the test agent produced an increase in the mutation frequency which was statistically different from the concurrent solvent control in the tests with and without S9 activation.

In the second test with S9 metabolic activation using a similar but narrower range of doses, no statistically significant effects of the test chemical were observed (see Table 4). A numerical increase in the mutant frequency was obtained at the 0.17% dose level but this increase was not statistically different from the concurrent solvent control value. The result with the 0.17% dose was not considered a significant effect because 1) the result was not statistically different from the concurrent control value; 2) no suggestion or evidence for a dose-related effect of the test chemical was observed in either of the two tests performed, 3) the value of the concurrent negative control using cell culture medium alone was essentially identical to the value obtained for the 0.17% dose and 4) similar random and unexplainable variations in the spontaneous mutation frequency are not unusual in the historical negative control data for this test system.

Table 1. Chinese Hamster Ovary (CHO) Mutation Assay: Determination of Cytotoxicity 24 Hours After Chemical Treatment With and Without Metabolic Activation.

Test Chemicals	Colonies / Plate*	Total # Colonies	% Survival Mean (±S.D.)	% of Solvent Control
Test Without S9 Activation
[NIAX^®Catalyst A-99]
0.30% v/v	Cytotoxic**	Cytotoxic**	< 1	< 1
0.25% v/v	40,29,25,24	118	29.5 (7.3)	25.7
0.20% v/v	94,84,80,96	354	88.5 (7.7)	77.1
0.15% v/v	93,126,108,102	429	107.2 (13.9)	93.5
0.10% v/v	96,104,92,106	398	99.5 (6.6)	86.7
0.09% v/v	90,93,99,91	373	93.2 (4.0)	81.3
0.08% v/v	88,77,97,84	346	86.5 (8.3)	75.4
CONTROLS
[Solvent - H2O]	125,110,104,120	459	114.8 (9.5)	100.0
Medium	102,91,99,94	386	96.5 (4.9)	84.1
EMS (200 µl/ml)	74,65,71,71	281	70.2 (3.8)	61.2
Test With S9 Activation
[NIAX^®Catalyst A-99]
0.20% v/v	Cytotoxic**	Cytotoxic**	< 1	< 1
0.15% v/v	64,79,82,74	299	74.8 (7.9)	72.9
0.10% v/v	121,88,117,97	423	105.8 (15.8)	103.2
0.09% v/v	115,94,108,78	395	98.8 (16.4)	96.3
0.08% v/v	105,98,80,91	374	93.5 (10.7)	91.2
CONTROLS
[Solvent - H2O]	104,91,106,109	410	102.5 (7.9)	100.0
Medium	111,89,125,120	445	111.2 (15.9)	108.5
DMN (100 µl/ml)	8,7,7,6	28	7.0 (0.8)	6.8

* 100 cells innoculated into each plate.

** Majority of cells were detached or lysed; survival could not be determined.

Abbreviations: S9 - rate liver homogenate; EMS - ethylmethanesulfonate; DMN - dimethylnitrosamine; S.D. - standard deviation.

Table 2. Results on Evaluation of Plating Efficiency and Mutant Induction Determined After Expression Period.

Test Chemicals	Plating Efficiency			Mutant Determination
	Colonies / Plate*	Mean Colonies / Plate (±S.D.)	% of Solvent Control	Mutant Colonies			Corrected†Mutation Frequency (X10^-6)
				Colonies / Plate**	Mean (±S.D.)	Total Colonies
Test Without S9 Activation
[NIAX^®Catalyst A-99]
0.25% v/v	78,100,82,83	85.8 (9.7)	83.3	0,0,0,0,0	0	0	0
0.20% v/v	97,88,103,97	96.2 (6.2)	93.4	1,1,0,0,0	0.4 (0.5)	2	2.1
0.15% v/v	95,95,107,88	96.2 (7.9)	93.4	2,1,1,1,0	1.0 (0.7)	5	5.2
0.10% v/v	84,74,66,84	77.0 (8.7)	74.8	0,0,0,0,0	0	0	0
0.09% v/v	84,112,111,98	101.2 (13.1)	98.3	0,0,0,0,0	0	0	0
0.08% v/v	104,96,76,86	90.5 (12.2)	87.9	1,0,0,0,0	0.2 (0.4)	1	1.1
CONTROLS
[Solvent - H2O]	105,110,111,86	103.0 (11.6)	100.0	1,1,0,0,0	0.4 (0.5)	2	1.9
Medium	C,131,134,141	135.3 (5.1)	131.4	0,0,0,0,0	0	0	0
EMS (200 µl/ml)	85,88,80,84	84.2 (3.3)	81.7	20,20,22,24,25	22.2 (2.3)	111	131.8^c
Test With S9 Activation
[NIAX^®Catalyst A-99]
0.15% v/v	73,48,49,49	54.8 (12.2)	57.6	1,1,0,0,0	0.4 (0.5)	2	3.7
0.10% v/v	98,75,94,107	93.5 (13.5)	98.2	0,0,1,1,1	0.6 (0.5)	3	3.2
0.09% v/v	111,95,75,111	98.2 (16.6)	103.2	0,1,2,2,4	1.8 (1.5)	9	9.2
0.08% v/v	109,95,115,115	108.5 (9.4)	114.0	1,0,0,0,0	0.2 (0.4)	1	0.9
CONTROLS
[Solvent - H2O]	100,99,99,83	95.2 (8.2)	100.0	1,2,3,4,6	3.2 (1.9)	16	16.8
Medium	92,85,90,80	86.8 (5.4)	91.2	0,0,0,0,0	0	0	0
DMN (100 µl/ml)	64,47,70,50	57.8 (11.0)	60.7	54,63,76,79,94	73.2 (15.4)	366	633.8^c

* 100 cells inoculated into each of 4 plates. C = Contaminated during incubation.

** 2 x 10^5 cells inoculated in each of 5 plates, (1 x 10^6 total cells).

† Mutant/10^6 clonable cells: total # mutant colonies divided by viable fraction.

Statistical significance above solvent control: c: p < 0.001

No superscript indicates p > 0.05. Data analyzed by Student's t-test.

Abbreviations: S9 - rat liver homogenate; EMS - ethylmethanesulfonate; DMN - dimethylnitrosamine; S.D. - standard deviation

Table 3. Determination of Cytotoxicity 24 Hours After Chemical Treatment with Metabolic Activation - Repeat Test

Test Chemicals	Colonies / Plate*	Total # Colonies	% Survival Mean (±S.D.)	% of Solvent Control
Test With S9 Activation
[NIAX^®Catalyst A-99]
0.18% v/v	86,56,76,65	283	70.8 (13.0)	78.0
0.17% v/v	96,90,94,83	363	90.8 (5.7)	100.0
0.16% v/v	89,87,76,100	352	88.0 (9.8)	97.0
CONTROLS
Solvent (H2O)	76,90,87,110	363	90.8 (14.2)	100.0
Medium	119,96,72,82	369	92.2 (20.4)	101.7
DMN (100 µl/ml)	5,8,9,12	34	8.5 (2.9)	9.4

*100 cells inoculated into each plate

Abbreviations: S9 - rat liver homogenate; DMN - dimethylnitrosamine; S.D. - standard deviation.

Table 4. Repeat Test With Metabolic Activation System Results on Evaluation of Plating Efficiency and Mutant Induction Determined After Expression Period.

Test Chemicals	Plating Efficiency			Mutant Determination
	Colonies / Plate*	Mean Colonies / Plate (±S.D.)	% of Solvent Control	Mutant Colonies			Corrected†Mutation Frequency (X10^-6)
				Colonies / Plate**	Mean (±S.D.)	Total Colonies**
Test With S9 Activation
[NIAX^®Catalyst A-99]
0.18% v/v	96,99,76,89	90.0 (10.2)	107.8	4,2,1,1,0	1.6 (1.5)	8	8.9
0.17% v/v	68,92,76,81	79.2 (10.0)	94.9	1,3,5,5,6	4.0 (2.0)	20	25.2
0.16% v/v	89,88,69,80	81.5 (9.3)	97.6	2,0,0,0,0	0.4 (0.9)	2	2.5
CONTROLS
Solvent (H2O)	97,78,86,73	83.5 (10.5)	100.0	3,2,2,1,0	1.6 (1.1)	8	9.6
Medium	71,88,81,77	79.2 (7.1)	94.9	6,4,4,4,0	3.6 (2.2)	18	22.7
DMN (100 µl/ml)	71,70,55,53	62.2 (9.6)	74.5	36,39,42,43,46	41.2 (3.8)	206	330.9^c

* 100 cells inoculated into each plate.

** 2 x 10^6 clonable cells: total # mutant colonies divided by viable fraction.

† Mutants/10^6 clonable cells: total # mutant colonies divided by viable fraction.

Statistical significance above solvent control: c: p < 0.001

No superscript indicateds p > 0.05. Data analyzed by Student's t-test.

Abbreviations: S9 - rat liver homogenate; DMN - dimethylnitrosamine; S.D. - standard deviation.

Conclusions:

NIAX Catalyst A-99 did not produce any statistically significant increases in the frequency of mutations of CHO cells at concentrations between 0.08 to 0.25% (by volume) in tests without an S9 metabolic activation system. With S9 activation, two tests, with either a similar or narrower range of concentrations as tested without S9, produced no indication of a dose-related, statistically significant effect of the treatements. NIAX Catalyst A-99 was not active in producing gene mutations in CHO cells in these studies.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

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 not conducted under GLP conditions.

Justification for type of information:

Following the read across strategy, this endpoint is covered by a key study performed with the structural analogue BDMAEE (N, N, N', N'-tetramethyl-2,2'-oxybis(ethylamine)). The justification for read across is attached in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

No reliable data for this endpoint is available with BAEE. Data generated with the analogue substance BDMAEE is used for endpoint coverage. NIAX Catalyst A-99 did not produce any statistically significant increases in the frequency of mutations of CHO cells at concentrations between 0.08 to 0.25% (by volume) in tests without an S9 metabolic activation system. With S9 activation, two tests, with either a similar or narrower range of concentrations as tested without S9, produced no indication of a dose-related, statistically significant effect of the treatements. NIAX Catalyst A-99 was not active in producing gene mutations in CHO cells in these studies. The same is assumed to be correct for BAEE.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

One in vivo micronucleus assay (in vivo cytogenicity test) with the read across source substance BDMAEE is available. The result of this study was negative.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991-05-28 to 1993-01-29

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The GLP study was conducted according to internal study methods (BRRC Project No. 91-15-18271) and in accordance with Organization for Economic Cooperation and Development Guidelines for Testing Chemicals Health Effects Test Guidelines, Office of Toxic Substances, U. S. Environmental Protection Agency (TSCA) 40 CFR Part 798.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

The study was conducted according to internal study methods (BRRC Project No. 91-15-18271) and in accordance with Organization for Economic Cooperation and Development Guidelines for Testing Chemicals Health Effects Test Guidelines, Office of Toxic Substances, U. S. Environmental Protection Agency (TSCA) 40 CFR Part 798.

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

- Name of test material (as cited in study report): N,N,N',N'-tetramethyl-2,2'-oxybis(ethylamine)
- Substance type: Pure actuve substance
- Physical state: Clear liquid
- Analytical purity: 96.2%
- Storage condition of test material: Stored at room temperature
- Lot/batch No.: Lot No. 84505, Sample No. 54-109
- Impurities (identity and concentrations): 0.5% DMEA, 0.08% 117 MW Amine, 1.7% 133 MW Dimethylamine, 0.06% 146 MW Amine, 0.03% 160 MW Amine, 0.9% DMAE- Dimethylaminoethyl ethanediamine (EDA), 0.15% 174 MW Amine (combined), 0.3% Other purities.
- Composition of test material, percentage of components: 96.2 Test substance, 0.5% DMEA, 0.08% 117 MW Amine, 1.7% 133 MW Dimethylamine, 0.06% 146 MW Amine, 0.03% 160 MW Amine, 0.9% DMAE- Dimethylaminoethyl ethanediamine (EDA), 0.15% 174 MW Amine (combined), 0.3% Other purities.

Species:

mouse

Strain:

Swiss Webster

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, (Portage, MI)
- Age at study initiation: Approximately 5 weeks old
- Weight at study initiation: 23.5 to 28.8 g for males and 19.7 to 22.6 g for females
- Assigned to test groups randomly: [Yes, under following basis: Following the pretest body weight, the animals were assigned to 3 treatment groups, a vehicle control group, and a positive control group using a nonstratified randomization procedure based on body weight. At the time of group assignment, only animals with body weights within two standard deviations of the population mean for each sex were included.]
- Fasting period before study: N/A
- Housing: Group housed in shoe-box type plastic cages (30.0 X 20.0 X 12.5 cm)
- Diet (e.g. ad libitum): Pelleted, certified AGWAY PROLAB Animal Diet Rat, Mouse, Hamster 3000 (Agway Inc.) was available d libitum
- Water (e.g. ad libitum): Tap water (Municipal Authority of Westmoreland County, Greensburg, PA) was available ad libitum
- Acclimation period: Approximately 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 4 deg C
- Humidity (%): 40-70%
- Air changes (per hr): N/A
- Photoperiod (hrs dark / hrs light): 12-hour photoperiod (approximately 0500 to 1700 hours for the light phase)

IN-LIFE DATES: From: 1991-05-27 To: 1991-06-07

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: Sterile, distilled, deionized water
- Justification for choice of solvent/vehicle: N/A
- Concentration of test material in vehicle: N/A
- Amount of vehicle (if gavage or dermal): 10 ml/kg
- Type and concentration of dispersant aid (if powder): N/A
- Lot/batch no. (if required): BRRC Sample Nos. 53-101A through U
- Purity: N/A

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Each test substance dosing solution was prepared by diluting the test substance in water. Concentrations were not adjusted for percent active ingredient of test substance. They were prepared on the day of treatment and were stored at room temperature prior to use. The TEM positive control dosing solution was prepared by dissolving it in ethanol then diluting it with sterile, distilled, deionized water. It was
prepared on the day of treatment and was stored at room temperature prior to use.

DIET PREPARATION
- Rate of preparation of diet (frequency): On the day of treatment
- Mixing appropriate amounts with (Type of food): N/A
- Storage temperature of food: N/A

- Justification for dose concentration: Based upon mortality data obtained in a range-finding study, the acute intraperitoneal LD50 for the combined sexes was calculated to be 181 mg/kg (no 95% confidence interval was calculated since none of the dose levels had partial survival). The doses for the definitive micronucleus assay were selected by the study director as approximately 25%, 50%, and 80% of the LD50 or 45, 90, and 145 mg/kg of the test substance.

Duration of treatment / exposure:

72 hours

Frequency of treatment:

Single dose

Post exposure period:

3 days

Dose / conc.:

45 mg/kg diet

Remarks:

nominal concentration

Dose / conc.:

90 mg/kg diet

Remarks:

nominal concentration

Dose / conc.:

145 mg/kg diet

Remarks:

nominal concentration

No. of animals per sex per dose:

5/sex/ treated with 45 or 90 mg/kg
8/sex/treated with 145 mg/kg
5/sex/treated with either the vehicle control (water, 10.0 ml/kg) or positive control (triethylenemelamine, 0.30 mg/kg)

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control: triethylenemelamine (Lot No. 84505, BRRC Sample No. 52-793)
- Justification for choice of positive control(s): Consistently produced positive responses in prior tests; Peripheral blood was collected 30 hours
after treatment.
- Route of administration: Single intraperitoneal injection
- Doses / concentrations: 0.30 mg/kg

Tissues and cell types examined:

Bone marrow, polychromatophilic erythrocytes (PCE) and normochromic erythrocytes (NCE).

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The test substance doses of approximately 25%, 50%, and 80% of the LD50, or 45, 90, and 145 mg/kg were selected by the Study Director and were based upon the results of the range-finding study that was a part of this experimental protocol.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Blood samples were collected approximately 30, 48 and 72 hours after dosing. Blood samples were collected from the positive control animals approximately 30 hr after dosing.

DETAILS OF SLIDE PREPARATION: Slides were coded with the randomly-assigned animal numbers and read without knowledge of the treatment group to prevent bias.

METHOD OF ANALYSIS: Blood samples of one to a few drops were collected by nicking the tail with a scalpel. One or two blood smears were made per animal per sampling time. Micronuclei in peripheral polychromatic erythrocytes were stained with Curres 8-66 Ciemsa diluted in phosphate buffer. Slides were coded with the randomly-assigned animal numbers and read without knowledge of the treatment group to prevent bias. The PCE:NCE ratio for approximately 1000 total cells will be calculated, recorded, and shown in the final report to provide an estimate of the cytotoxicity of the test agent. Excessive cytotoxicity in this test system will be defined by a PCE:NCE ratio of 0.01 or lower. Mice with an excessive reduction in the PCE:NCE ratio will be identified in the raw data and final report.

OTHER: N/A

Evaluation criteria:

One to a few drops of blood were collected from the tail by nicking it with a scalpel. One or two blood smear slides were prepared/animal/sampling time. Micronuclei in peripheral blood polychromatophilic erythrocytes were stained with Gurr's R-66 Giemsa diluted in phosphate buffer. Slides were coded with the randomly-assigned animal number and read without knowledge of treatment group to prevent bias. Slides were identified by BRRC project number, animal number and sampling interval.

The PCE:NCE ratio for a total of 1000 cells for each animal was calculated to provide an estimate of test substance cytotoxicity. A minimum of 1000 PCE for each animal were scored for the presence of micronuclei unless the cytotoxicity of the test substance prevented this.

Statistics:

Data were statistically evaluated using the Mann-Whitney U test. All statistical analyses were performed using BMDP Statistical Software. For all statistical tests, the probability value of < 0.05 (two-tailed) was used as the critical level of significance. Various models of calculators, computers, and computer programs may have been used to analyze data for this study. Since various models round or truncate numbers differently, values in some tables may differ slightly from those in other tables or from independently calculated data. The integrity of the study and interpretation of the data were unaffected by these differences.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 32, 64, 128,256 and 512 mg/kg
- Solubility: N/A
- Clinical signs of toxicity in test animals: The aniamls were observed for signs of toxic and/or pharmacological effects for 72 hr after dosing. Blood was taken from the tail vein at 48 hr and 72 hr after dosing from the 128 mg/kg and water control animals, and was used for determining the ratio of polychromatophilic erythrocytes (PCE) to normochromic erythrocytes (NCE). Body weights were measured before dosing and before sacrifice on the fourth day after dosing (approximately 72 hr postdosing), 5 of 5 males and females were found dead in the 256 and 512 mg/kg dose groups after 72 hours. By the end of the 72 hr observation period, the body weights of the 128 mg/kg males were statistically significantly reduced; whilst those of the females of this dosage group were also reduced, this was not statistically significant. By 72 hr, body weight gains for both males and females were reduced.
- Evidence of cytotoxicity in tissue analyzed: 48 hr sample for 0 mg/kg= 27.6 +/- 2.1 PCE/NCE (male) and 38.0 +/- 8.0 PCE/NCE (female); 72 hr sample for 0 mg/kg= 27.0 +/- 2.6 PCE/NC (male) and 33.0 +/- 6.2 PCE/NCE; 48 hr for 128 mg/kg= 28.6 +/- 5.0 (male) and 35.8 +/- 9.6 (female); 72 hr for 128 mg/kg= 26.4 +/- 5.6 (male) and 29.4 +/- 8.8 (female). The findings showed no evidence for bone marrow toxicity from the test substance given ip at a dosage of 128 mg/kg.

- Rationale for exposure: N/A
- Harvest times: 48 and 72 hours
- High dose with and without activation: N/A
- Other: The control group received ip distilled water (10 ml/kg).

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): N/A
- Induction of micronuclei (for Micronucleus assay): Blood samples of one to a few drops were collected by nicking the tail with a scalpel. One or two blood smears were made per animal per sampling time. Micronuclei in peripheral polychromatic erythrocytes were stained with Curres 8-66 Ciemsa diluted in phosphate buffer. Slides were coded with the randomly-assigned animal numbers and read without knowledge of the treatment group to prevent bias. The PCE:NCE ratio for approximately 1000 total cells will be calculated, recorded, and shown in the final report to provide an estimate of the cytotoxicity of the test agent. Excessive cytotoxicity in this test system will be defined by a PCE:NCE ratio of 0.01 or lower. Mice with an excessive reduction in the PCE:NCE ratio will be identified in the raw data and final report.
- Ratio of PCE/NCE (for Micronucleus assay): The ratio of PCE/NCE was determined to assess any bone marrow cytotoxicity, and the number of micronucleated erythrocytes was counted and expressed as the proportion of polychromatophilic erythrocytes with micronuclei. There were no significant decreases in PCE/NCE ratios at any dosage, indicating an absence of bone marrow cytotoxicity. There were no significant changes in the incidence of micronucleated polychromatophils with either sex at any dosage, when compared with the water-alone controls. The positive control, TEM, produced highly statistically significant increases in micronucleated polychromatophils with evidence of cytotoxicity.
- Appropriateness of dose levels and route: Yes
- Statistical evaluation: See Statistics section above

The above dosage-mortality data allowed the calculation of a 72 hr-ip LD50 of 181 mg kg- for both male and female Swiss-Webster mice. By the end of the 72 hr observation period, the body weights of the 128 mg kg males were statistically significantly reduced; whilst those of the females of this dosage group were also reduced, this was not statistically significant. By 72 hr, body weight gains for both males and females were reduced.

Results of the mouse peripheral blood micronucleus test with the test substance given by intraperitoneal injection

Group	Dosage	Sex	PCE/NCE (% Controls)^a			Micronuclei (% PCE with MN)
			30 hr	48 hr	72 hr	30 hr	48 hr	72 hr
Water^b	10 ml/kg	Male				0.40	0.30	0.44
		Female	-	-		0.24	0.12	0.24
Test substance	45 ml/kg	Male	113.0	126.4	101.5	0.46	0.26	0.34
		Female	106.4	107.8	104.2	0.28	0.3	0.16
	90 ml/kg	Male	111.6	114.4	112.5	0.34	0.20	0.34
		Female	88.5	97.4	109.7	0.28	0.02	0.36
	145 ml/kg	Male	103.4	116.8	105.9	0.48	0.26	0.42
		Female	91.1	94.2	113.2	0.20	0.16	0.24
Positive control^c	0.3 ml/kg	Male	46.6		-	2.90
		Female	39.5			2.10

^ARatio of polychromatophilic erythrocytes to normochromic erythrocytes as % of control

(water) values.

^BControl group.

^CTEM = triethylenemelamine positive control (30 hr sample only).

Conclusions:

The test substance did not produce significant, treatment-related increases in the incidence of micronucleated polychromatophilic erythrocytes among male or female Swiss-Webster mice assessed at concentrations of 45, 90, or 145 mg/kg did not produce significant, treatment-related increases in the incidence of micronucleated polychromatophilic erythrocytes among male or female Swiss-Webster mice assessed at 30, 48, or 72 hours after treatment with a single dose by intraperitoneal injection. Therefore, the test substance was not considered to be an inducer of micronuclei in male or female Swiss-Webster mice under the conditions of this in vivo assay.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

Following the read across strategy, this endpoint is covered by a key study performed with the structural analogue BDMAEE (N, N, N', N'-tetramethyl-2,2'-oxybis(ethylamine)). The justification for read across is attached in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

No reliable data with BAEE is available for this endpoint. Data generated with the analogue substance BDMAEE is used for endpoint coverage. BDMAEE did not produce significant, treatment-related increases in the incidence of micronucleated polychromatophilic erythrocytes among male or female Swiss-Webster mice assessed at concentrations of 45, 90, or 145 mg/kg did not produce significant, treatment-related increases in the incidence of micronucleated polychromatophilic erythrocytes among male or female Swiss-Webster mice assessed at 30, 48, or 72 hours after treatment with a single dose by intraperitoneal injection. Therefore, BDMAEE was not considered to be an inducer of micronuclei in male or female Swiss-Webster mice under the conditions of this in vivo assay. The same is assumed to be correct for the test substance BAEE.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity: in vitro:

Bacterial reverse mutation assay (e.g. Ames test):

The results of an Ames Assay with the test substance (according to OECD Guideline 471) were negative in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 and E. coli strain WP2 uvrA , both with and without metabolic activation at following doses:

Experiment 1 (initial toxicity - mutation assay): 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg/plate (all strains tested)

Experiment 2: retest of tester strain WP2 uvrA in presence of S9 activation due to unacceptable vehicle control value in experiment 1: 50, 150, 500, 1500 and 5000 µg/plate

Experiment 3 (confirmatory mutagenicity assay): 50, 150, 500, 1500 and 5000 µg/plate

Experiment 4: retest of tester strain TA1537 in the presence of S9 due to numerous intermediate colonies that interfered with evaluation of the assay plates: 50, 150, 500, 1500 and 5000 µg per plate.

In vitro mammalian cell gene mutation test:

In a study conducted equivalent to OECD Guideline 476, the read-across substance BDMAEE did not produce any statistically significant increases in the frequency of mutations of CHO cells at concentrations between 0.08 to 0.25% (by volume) in tests without an S9 metabolic activation system. With S9 activation, two tests, with either a similar or narrower range of concentrations as tested without S9, produced no indication of a dose-related, statistically significant effect of the treatments. BDMAEE was not active in producing gene mutations in CHO cells in these studies.

In vitro chromosome aberration test:

No in vitro cytogenicity study in mammalian cells is necessary as adequate data from an in vivo cytogenicity test with the read-across substance BDMAEE is available (REACH Annex VIII, Section 8.4.2 column 2).

Genetic toxicity: in vivo:

Mammalian erythrocyte micronucleus test:

In a study conducted equivalent to OECD Guideline 474, with the read-across substance BDMAEE did not produce significant, treatment-related increases in the incidence of micronucleated polychromatophilic erythrocytes among male or female Swiss-Webster mice assessed at concentrations of 45, 90, or 145 mg/kg did not produce significant, treatment-related increases in the incidence of micronucleated polychromatophilic erythrocytes among male or female Swiss-Webster mice assessed at 30, 48, or 72 hours after treatment with a single dose by intraperitoneal injection. Therefore, the test substance was not considered to be an inducer of micronuclei in male or female Swiss-Webster mice under the conditions of this in vivo assay

Justification for classification or non-classification

Based on the available data and according to the criteria of the CLP Regulation (EC) 1272/2008, the test substance should not be classified for mutagenicity.