2,2'-oxydi(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:

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-oxydi(ethylamine)

EC Number:

220-395-5

EC Name:

2,2'-oxydi(ethylamine)

Cas Number:

2752-17-2

Molecular formula:

C4H12N2O

IUPAC Name:

2-(2-aminoethoxy)ethan-1-amine

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

Method

Target gene:

Histidine and tryptophan locus

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

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.

Controls

Controls 1

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

Controls 2

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

Controls 3

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

Controls 4

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

Controls 5

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.

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:

no cytotoxicity

Remarks:

Neither precipitate nor toxicity was observed

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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

Applicant's summary and conclusion

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.