2-[(3-aminopropyl)methylamino]ethanol

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:

2017-06-06 to 2017-06-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

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: ISO/IEC 17025:2005

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-[(3-aminopropyl)methylamino]ethanol

EC Number:

255-615-9

EC Name:

2-[(3-aminopropyl)methylamino]ethanol

Cas Number:

41999-70-6

Molecular formula:

C6H16N2O

IUPAC Name:

2-[(3-aminopropyl)(methyl)amino]ethan-1-ol

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: PFW170183
- Expiration date of the lot/batch: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light

- purity: 98.83%
- molecular weight: 132.2 g/mol
- appearance: clear colorless liquid

Method

Target gene:

Histidine locus (Salmonella strains) and tryptophan locus (E. coli strain)

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):

- TA98 and TA1537 are reverted from histidine dependence to histidine independence by framashift mutagens
- TA 1535 is reverted by mutagens that cause basepair substitutions
- TA100 is reverted by mutagens that cause both framashift and basepair substitutions mutations.
- E. coli is sensitive to basepair substitution mutationsrather than frameshift mutations

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Initial Toxicity-Mutation Assay: 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate with and without S9-mix;
Confirmatory Mutagenicity Assay: 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate with and without S9-mix;

Since the test item formed a clear solution in sterile water for injection at approx. 50 mg/mL, 5000 µg/plate was selected as the maximum concentration in the initial toxicity-mutation assay. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Water was the vehicle 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 a concentration of approximately 50 mg/mL in the solubility test conducted at the testing facility.
- Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under filtered light.

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

Remarks:

With S9: 1.0 μg/plate for TA98, TA1535 ; 2.0 μg/plate for TA100, TA1537; 15 μg/plate for WP2 uvrA

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 S9: 1.0 μg/plate 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 S9: 1.0 μg/plate for TA100, 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 S9: 75 μg/plate 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 S9: 1000 μg/plate for WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Initial toxicity-mutation assay: used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone, positive controls and eight dose levels of the test substance, in duplicate, in the presence and absence of metabolic activation. Dose levels for the confirmatory mutagenicity assay were based uppon post-treatment toxicity.
- Confirmatory mutagenicity assay: was used to evaluate and confirm the mutagenic potential of the test substance. TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone, positive controls and six dose levels of the test substance, in triplicate, in the presence and absence of metabolic activation.
-One half (0.5) milliliter of S9 or Sham mix, 100 µL of tester strain (cells seeded) and 100 µL of vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45±2°C. When plating the positive controls, the test article aliquot was replaced by a 50.0 µL aliquot of appropriate positive control. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay had solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2 8C until colony counting could be conducted.

DURATION
- Exposure duration: 48 to 72 hours
- Selection time (if incubation with a selection agent): 48 to 72 hours (simultaneous with exposure)

SELECTION AGENT (mutation assays): Histidine (S. typhimurium) or Tryptophan (E. coli)

NUMBER OF REPLICATIONS:
Initial toxicity-mutation assay: duplicate
Confirmatory mutagenicity assay: 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 each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article as specified below:
- Strains TA1535 and TA1537
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
- Strains TA98, TA100 and WP2 uvrA
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
An equivocal response is a biologically relevant increase in a 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:

No formal hypothesis testing was done.
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, other: TA98, TA100, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Soluble at 50 mg/mL
- Precipitation: no precipitation has been observed
- Other: sterility results: No contaminant colonies were observed on the sterility plates for the vehicle control, the test article dilutions or the S9 and Sham mixes.

RANGE-FINDING/SCREENING STUDIES:
The initial toxicity-mutation assay was used to establish the dose range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone, positive controls and eight dose levels of the test article, in duplicate, in the presence and absence of Aroclor induced rat liver S9. Neither precipitate nor toxicity was observed. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Based upon the results of the initial toxicity-mutation assay, the dose levels selected for the confirmatory mutagenicity assay were 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate. No precipitate was observed. Toxicity as reduction in revertant count was observed at 5000µg per plate with tester strain TA1535 in the presence of S9 activation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: all positive controls exhibited at least 3-fold increases in the number of revertants
- Negative (solvent/vehicle) historical control data: the number of revertants was within the characteristic ranges for all vehicle controls
All criteria for a valid study were met as described in the protocol.

Applicant's summary and conclusion

Conclusions:

All criteria for a valid study were met. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of the study, the test item did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor induced rat liver S9.