2-(2,4-diaminophenoxy)ethanol dihydrochloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study performed according to OECD guideline 471 and in compliance to GLP.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Reference 2

Reference Type:

other: Published secondary source

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(2,4-diaminophenoxy)ethanol dihydrochloride

EC Number:

266-357-1

EC Name:

2-(2,4-diaminophenoxy)ethanol dihydrochloride

Cas Number:

66422-95-5

Molecular formula:

C8H12N2O2.2ClH

IUPAC Name:

2-(2,4-diaminophenoxy)ethan-1-ol dihydrochloride

Details on test material:

Test item : 2,4-Diaminophenoxyethanol HCl
EC number : 266-357-1
Batch number : 0120022
Purity : >99.5%

Method

Target gene:

Histidine locus.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: see below

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: see below

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 fraction from rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Range finder experiment
1.6, 8, 40, 200, 1000, 5000 ug/plate with and without S9 mix for strain TA100

Mutation experiment 1
1.6, 8, 40, 200, 1000, 5000 ug/plate with and without S9 mix for strains TA98, TA102, TA1535, TA1537

Mutation experiment 2
1.3, 3.3 ug/plate with and without S9 mix for strain TA102
8.2 ug/plate with and without S9 mix for strains TA98, TA102
20.5, 51.2, 128, 320, 800, 2000, 5000 ug/plate with and without S9 mix for strains TA98, TA100, TA102, TA1535, TA1537

Vehicle / solvent:

Purified water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

(used for strain TA98)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

(used for strains TA100, TA1535)

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

(used for strain TA1537)

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: glutaraldehyde

Remarks:

(used for strain TA102)

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

(used for strain TA98)

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

(used for strains TA100, TA1535, TA1537)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 1 hour (experiment 2)
- Exposure duration: 48 hours
- Expression time (cells in growth medium): N/A
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A

SELECTION AGENT (mutation assays): L-histidine
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): N/A

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: N/A

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test substance was measured by a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

Evaluation criteria:

The test substance was considered to be mutagenic if the assay was valid (see below), Dunnett's test gave a significant response (p<=0.01) and the data set showed a significant dose correlation, the positive responses were reproducible.

The assay was considered valid if the mean negative control counts fell within the normal ranges, the positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains and an active S9 preparation, no more than 5% of the plates were lost through contamination or some other unforeseen event.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA100, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: TA100 only

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium, other: TA98, TA102

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium, other: TA98, TA102

Metabolic activation:

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 4

Species / strain:

S. typhimurium, other: TA100, TA1535, TA1537 (experiment 2)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in presence of S9 at highest four test doses

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium, other: TA102 (experiment 2)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at highest two test doses

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium, other: TA102 (experiment 2)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at highest two test doses

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Species / strain:

S. typhimurium, other: TA98 (experiment 2)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at highest four test doses

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 8

Species / strain:

S. typhimurium, other: TA98 (experiment 2)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Toxicity, Solubility and Dose Selection

An initial toxicity range-finder experiment was carried out in strain TA100 only, using final test substance concentrations at 1.6, 8, 40, 200, 1000 and 5000 ug/plate plus solvent and positive controls. No clear evidence of toxicity was observed following these treatments and these results were used to comprise the TA100 mutagenicity data for experiment 1.

Experiment 1 treatments of the remaining test strains retained the same test doses as employed for the range-finder experiment treatments. No clear evidence of toxicity was observed in any of the test strains.

Experiment 2 treatments of all the test strains were performed with a maximum test dose of 5000 ug/plate. Narrowed dose ranges were employed. In addition, all treatments in the presence of S9 were further modified by the inclusion of a pre-incubation step in an attempt to increase the range that could be detected using this assay system. Following pre-incubation treatments, evidence of toxicity in the form of a slight thinning of the background bacterial lawn was observed for strain TA102 at the highest two test doses in the absence and presence of S9. Evidence of toxicity was also observed following plate incorporation treatments in the presence of S9 at the highest four test doses.

The test substance was completely soluble in the aqueous assay system at all concentrations treated in each of the experiments performed.

Mutation

Following experiment 1 treatments statistically significant increases in revertants were observed for strains TA98 and TA102. in the presence of S9. Following experiment 2 plate incorporation treatments of these strains statistically significant increases were observed for strain TA98. This increase was dose related and reproducible in experiment 1 and was therefore considered evidence of test substance mutagenic activity in TA98 in the presence of S9. No statistically significant increases in revertant numbers were observed for strain TA102. Therefore the increases in revertants observed in experiment 1 were not reproducible and were considered to be due to a chance event and not indicative of test substance mutagenic activity in TA102.

Following experiment 2 pre-incubation treatments statistically significant increases in revertants were observed for strains TA98 and TA102 in the presence of S9. The increase observed for TA102 occurred at a low dose level, was small in magnitude and therefore was considered to be due to a chance event and not indicative of mutagenic activity in this strain. the increase in revertants observed for strain TA98 was observed at an intermediate and high dose level and showed some evidence of a dose response. Therefore this is considered supporting evidence of test substance mutagenicity in strain TA98 in the presence of S9.

No other significant, dose related and reproducible increases in revertants were observed for any other strain tested.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation

2,4-Diaminophenoxyethanol HCL induced mutations in Salmonella typhimurium strain TA98 in the presence of S9 when tested under the conditions of this study.

Executive summary:

2,4 -Diaminophenoxyethanol dihydrochloride was investigated for the induction of gene mutations in Salmonella typhimurium. Liver S9 fraction from rats induced with Aroclor 1254 was used as the exogenous metabolic activation system. Negative and positive controls were in accordance with the OECD guideline. No clear evidence of toxicity was observed in any of the test strains in the first experiment (1.6 - 5000 ug/plate), but the data were considered to be acceptable for mutation assessment. A statistically significant increase in revertants was observed in strains TA98 and TA102 in the presence of S9 mix. In the second experiment, different dose intervals were used for strains TA100, TA1535 and TA1537 (20.48 - 5000 ug/plate), strain TA98 (8.2 - 5000 ug/plate) and strain TA102 (1.3 - 5000 ug/plate); in the presence of metabolic activation a pre-incubation step was used. For strains TA98 and TA102 in the presence of S9, plate-incorporation treatments were additionally included to assess the reproducibility of increases in revertants observed in experiment 1. Following these treatments, evidence of toxicity was observed in strain TA102 only in the presence of S9 for pre-incubation and plate incorporation treatments. A statistically significant and dose-related increase in revertants was observed for TA98 in the presence of S9mix also in the second experiment. As an increase was not observed for TA102 in the second experiment. 2,4-Diaminophenoxyethanol dihydrochloride induced mutations in Salmonella typhimurium strain TA98 in the presence of S9 when tested under the conditions of this study.