Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
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:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[bis(2-ethylhexyl)amino]ethanol
EC Number:
202-912-6
EC Name:
2-[bis(2-ethylhexyl)amino]ethanol
Cas Number:
101-07-5
Molecular formula:
C18H39NO
IUPAC Name:
2-[bis(2-ethylhexyl)amino]ethan-1-ol
Test material form:
liquid

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
In the initial toxicity-mutation assay (B1), the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate
In the retest of initial toxicity-mutation assay (B2) with tester strains TA100 and TA1535 in the presence of S9 activation and with TA1535 in the absence of S9 activation, the dose levels tested were 0.150, 0.500, 1.50, 5.00, 15.0, 50.0, 150, 500 and 1500 µg per plate.
In the confirmatory mutagenicity assay (B3), the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate for tester strain TA98, TA1537 and WP2 uvrA and 0.150, 0.500, 1.50, 5.00, 15.0, 50.0, 150, 500 and 1500 µg per plate for tester strain TA100 and TA1535.
toxicity and precipitation was the basis for the dose setting.
Vehicle / solvent:
DMSO
supplied by Sigma Aldrich, 99.95% pure
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-Aminoanthracine

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Sterility Results

No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.

Tester Strain Titer Results

Experiment

Tester Strain

TA98

TA100

TA1535

TA1537

WP2uvrA

Titer Value (x 109cells per mL)

B1

1.4

1.5

1.5

2.8

3.8

B2

-

1.3

2.1

-

-

B3

2.2

1.3

1.9

3.2

3.6

Initial Toxicity-Mutation Assays

The results of the initial toxicity-mutation assay conducted at dose levels of 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate in DMSO are presented inTables 1and2. The maximum dose of 5000 µg per plate was achieved using a concentration of 50.0 mg/mL and a 100 µL plating aliquot.

Precipitate was observed at 5000 µg per plate with all conditions. Toxicity was observed beginning at 15.0, 50.0, 500, 1500 or at 5000 µg per plate as indicated in the following table:

Tester Strains

Without metabolic activation

(µg per plate)

With metabolic activation

(µg per plate)

Precipitate

Toxicity

Precipitate

Toxicity

TA98

5000

-

5000

-

TA100

5000

≥ 50.0

5000

≥ 500

TA1535

5000

≥ 15.0

--

-

TA1537

5000

5000a

5000

-

WP2uvrA

5000

1500a

5000

5000a

aToxicity was observed as a reduction in revertant counts.

 

However, the following test conditions were not evaluated, due to the observations indicated below, and were retested:

 

· excessive toxicity was observed with tester strain TA1535 in the absence of S9 activation,

· the vehicle controls for tester strain TA100 in the presence of S9 activation were outside of the acceptable historical control limits, and

· tester strain contamination was observed with TA1535 in the presence of S9 activation.

 

No positive mutagenic responses were observed with any of the tester strains that provided valid results in either the presence or absence of S9 activation.  Mutagenicity was not evaluated in the initial toxicity-mutation assay for tester strains TA100 and TA1535 in the presence of S9 activation and TA1535 in the absence of S9 activation, which were retested as indicated above.

In the retest of initial toxicity-mutation assay (B2) with tester strains TA100 and TA1535 in the presence of S9 activation and with TA1535 in the absence of S9 activation, the dose levels tested were 0.150, 0.500, 1.50, 5.00, 15.0, 50.0, 150, 500 and 1500 µg per plate. 

No precipitate was observed. Toxicity was observed beginning at 5.00 and 500 µg per plate. 

No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

Confirmatory Mutagenicity Assay

Based upon the results of the initial toxicity-mutation assay, the dose levels selected for the confirmatory mutagenicity assay were as indicated in the following table:

Tester Strains

Activation Condition

Dose Levels (µg per plate)

TA98

With and without

15.0, 50.0, 150, 500, 1500 and 5000

TA100

0.150, 0.500, 1.50, 5.00, 15.0, 50.0, 150, 500, 1500

TA1535

0.150, 0.500, 1.50, 5.00, 15.0, 50.0, 150, 500, 1500

TA1537

15.0, 50.0, 150, 500, 1500 and 5000

WP2uvrA

15.0, 50.0, 150, 500, 1500 and 5000

Precipitate was observed at 5000 µg per plate with most conditions. Toxicity was observed beginning at 5.00, 15.0, 500, 1500 or at 5000 µg per plate as indicated in the following table:

Tester Strains

Without metabolic activation

(µg per plate)

With metabolic activation

(µg per plate)

Precipitate

Toxicity

Precipitate

Toxicity

TA98

5000

-

5000

-

TA100

-

≥ 15.0

-

≥ 500

TA1535

-

≥ 5.00

-

≥ 500

TA1537

5000

5000a

5000

-

WP2uvrA

5000

1500a

5000

1500a

aToxicity was observed as a reduction in revertant counts.

No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

Applicant's summary and conclusion

Conclusions:
All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, 2-[Bis(2-ethylhexyl)amino]ethanol 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.

Executive summary:

The test substance, 2-[Bis(2-ethylhexyl)amino]ethanol, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains ofSalmonella typhimuriumand at the tryptophan locus ofEscherichia colistrain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

 

In the initial toxicity-mutation assay (B1), the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate.  Precipitate was observed at 5000 µg per plate with all conditions.  Toxicity was observed beginning at 15.0, 50.0, 500, 1500 or at 5000 µg per plate with several conditions.  However, the following test conditions were not evaluated, due to the observations indicated below, and were retested:

· excessive toxicity was observed with tester strain TA1535 in the absence of S9 activation,

· the vehicle controls for tester strain TA100 in the presence of S9 activation were outside of the acceptable historical control limits, and

· tester strain contamination was observed with TA1535 in the presence of S9 activation.

No positive mutagenic responses were observed with any of the tester strains that provided valid results in either the presence or absence of S9 activation.  Mutagenicity was not evaluated in the initial toxicity-mutation assay for tester strains TA100 and TA1535 in the presence of S9 activation and TA1535 in the absence of S9 activation, which were retested as indicated above.

In the retest of initial toxicity-mutation assay (B2) with tester strains TA100 and TA1535 in the presence of S9 activation and with TA1535 in the absence of S9 activation, the dose levels tested were 0.150, 0.500, 1.50, 5.00, 15.0, 50.0, 150, 500 and 1500 µg per plate. No precipitate was observed. Toxicity was observed beginning at 5.00 and 500 µg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Based upon the initial toxicity-mutation and the retest of the initial toxicity-mutation results, the maximum dose tested in the confirmatory mutagenicity assay was 5000 µg per plate for tester strain TA98, TA1537 and WP2 uvrA and 1500 µg per plate for tester strain TA100 and TA1535.

In the confirmatory mutagenicity assay (B3), the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate for tester strain TA98, TA1537 and WP2 uvrA and 0.150, 0.500, 1.50, 5.00, 15.0, 50.0, 150, 500 and 1500 µg per plate for tester strain TA100 and TA1535. Precipitate was observed at 5000 µg per plate with most conditions. Toxicity was observed beginning at 5.00, 15.0, 500, 1500 or at 5000 µg per plate with most conditions. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

These results indicate 2-[Bis(2-ethylhexyl)amino]ethanol was negative for the ability to induce reverse mutations at selected loci of several strains ofSalmonella typhimuriumand at the tryptophan locus ofEscherichia colistrain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.