2-hydroxyethanesulphonic acid, compound with 4,4'-[hexane-1,6-diylbis(oxy)]bis[benzenecarboxamidine] (2:1)

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:

october-november 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxyethanesulphonic acid, compound with 4,4'-[hexane-1,6-diylbis(oxy)]bis[benzenecarboxamidine] (2:1)

EC Number:

211-533-5

EC Name:

2-hydroxyethanesulphonic acid, compound with 4,4'-[hexane-1,6-diylbis(oxy)]bis[benzenecarboxamidine] (2:1)

Cas Number:

659-40-5

Molecular formula:

C20H26N4O2.2C2H6O4S

IUPAC Name:

4-[6-(4-carbamimidoylphenoxy)hexoxy]benzenecarboximidamide;2-hydroxyethanesulfonic acid

Test material form:

solid: particulate/powder

Details on test material:

Batch identification: 00085890070
Date of production: 05 Oct 2011

Method

Target gene:

base pair substitutions

Species / strain

Species / strain / cell type:

E. coli WP2 uvr A

Test concentrations with justification for top dose:

In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate are generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose will be tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation.

Vehicle / solvent:

Due to the good solubility of the test substance in ultrapure water, ultrapure water was used as vehicle.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

other: 2-aminoanthracene

Details on test system and experimental conditions:

TEST SYSTEM
For testing, a deep-frozen (-70°C to -80°C) bacterial culture (E. coli WP2 uvrA) is thawed at room temperature, and 0.1 mL of this bacterial suspension is inoculated in nutrient broth solution (8 g/L Difco nutrient broth + 5 g/L NaCl) and incubated in the shaking water bath at 37°C for about 12 - 16 hours. As a rule, a germ density of ≥ 108 bacteria/mL is reached. This culture grown overnight is kept in iced water from the beginning of the experiment until the end in order to prevent further growth.
The use of the strain mentioned is in accordance with the current scientific recommendations for the conduct of this assay.
The Escherichia coli strain was obtained from Merck KGaA, Darmstadt, Germany (09 Sep 1991).

Rationale for test conditions:

Scope of tests and test conditions
1st Experiment
Strain: E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1 000; 2 500 and 5 000 μg/plate
Type of test: Standard plate test with and without S9 mix
Number of plates: 3 test plates per dose or per control

2nd Experiment
Strain: E. coli WP2 uvrA
Doses: 0; 1; 3.3; 10; 33; 100 and 333 μg/plate
Type of test: Standard plate test with and without S9 mix
Number of plates: 3 test plates per dose or per control
Reason: Bacteriotoxicity was observed in the standard plate test.

3rd Experiment
Strains: E. coli WP2 uvrA
Doses: 0; 1; 3.3; 10; 33; 100 and 333 μg/plate
Type of test: Preincubation test with and without S9 mix
Number of plates: 3 test plates per dose or per control
Reason: No mutagenicity was observed in the standard plate test.

Evaluation criteria:

Acceptance criteria
Generally, the experiment is considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 109 cells per mL were used. For approval
the titer of viable bacteria was ≥ 108 colonies per mL.

Assessment criteria
The test substance is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in the tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
• The number of revertants are within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: bacteriotoxicity

Remarks:

see additional information

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TOXICITY
Strong bacteriotoxicity (reduced trp- background growth, decrease in the number of trp+ revertants, reduction in the titer) was observed in the standard plate test depending on the test conditions from about 100 μg/plate onward.
In the preincubation assay bacteriotoxicity (decrease in the number of trp+ revertants) was observed depending on the test conditions from about 100 μg/plate onward.

Any other information on results incl. tables

STRAIN: E. coli WP2 uvrA

DOSE RANGE: 1 μg - 5 000 μg/plate (SPT)

1 μg - 333 μg/plate (PIT)

TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

SOLUBILITY: No Precipitation of the test substance was found at with and without S9 mix.

TOXICITY: Strong bacteriotoxicity observed from about 100 μg/plate

onward (for details see item 4.2.).

Applicant's summary and conclusion

Conclusions:

Thus, under the experimental conditions chosen here, it is concluded that Hexamidine diidethionate is not a mutagenic test substance in the Escherichia coli reverse mutation test in the absence and the presence of metabolic activation.

Executive summary:

According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in three experiments carried out independently of each other (standard plate test and preincubation assay).

Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.

In this study with and without S9 mix, the number of revertant colonies in the negative controls was within or nearby the range of the historical negative control data.

In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control

data or above.