Chlorhexidine 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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

Chlorhexidine dihydrochloride

EC Number:

223-026-6

EC Name:

Chlorhexidine dihydrochloride

Cas Number:

3697-42-5

Molecular formula:

C22H30Cl2N10.2ClH

IUPAC Name:

chlorhexidine dihydrochloride

Test material form:

solid

Details on test material:

- Test substance: Chlorhexidine dihydrochloride
- Appearance/Colour: Powder, white to yellowish
- Analytical purity: 99.4%
- Purity test date: 2001-07-27
- Lot/batch No.: A-010019
- Stability: 5 years, dry and cool (<25°C) storage

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (Aroclor 1254-induced rat liver)

Test concentrations with justification for top dose:

Plate incorporation test / Preincubation test: 0, 0.01, 0.0316, 0.1, 0.316 and 1 ug/plate (concentrations are referred to the active ingredient).
The top concentration was chosen based on a preliminary test in TA 100 with 10 concentrations ranging from 0.316 - 5000 ug/plate, where pronounced to complete cytotoxicity was observed at >=1 μg/plate.

Vehicle / solvent:

The test substance was dissolved in water and added to the incubation assay.

Controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: without metabolic activation: sodium azide ; 2-nitrofluorene; 9- aminoacridine; methylmethanesulfonate; with metabolic activation: cyclophos phamide; 2-anthraceneamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation and preincubation
Preincubation period: Plate incorporation test: none; Preincubation test: 60 min at 37 °C
Plates: 3 per concentration; 2 independent experiments

Evaluation criteria:

Number of revertants is significantly increased compared to solvent controls to at least 2-fold (TA 98,
TA 100, TA 102) and 3-fold (TA 1535, TA 1537) in both independent experiments

Statistics:

U-test according to Mann & Whitney

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in preincubation test cytotxicity at 1 µg/plate without metabolic activation

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in plate incorporation test cytotxicity at 1 µg/plate without metabolic activation; in preincubation test cytotxicity at 1 µg/plate without metabolic activation

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in plate incorporation test cytotxicity at 1 µg/plate without metabolic activation; in preincubation test cytotxicity at 1 µg/plate without metabolic activation

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in preincubation test cytotxicity at 1 µg/plate with metabolic activation

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In a preliminary test ranging from 0.316 to 5000 µg/plate were tested in TA 100. Pronounced cytotoxicity was observed from 1µg/plate onwards.

Applicant's summary and conclusion

Conclusions:

No evidence for mutagenic activity in the Salmonella typhimurium assay.

Executive summary:

A mutagenicity study (reverse mutation assay) in Salmonella typhimurium according to OECD guideline 471 with plate incorporation test and preincubation test each with and without metabolic activation by S9 mix from Arochlor 1254-induced rat liver is reported. The concentration of the test substance was based on the observations of a preliminary test with strain TA 100 in which pronounced to complete cytotoxicity was observed at >= 1 μg/plate. Under the conditions of the assay where positive controls exerted the expected mutagenic effects, chlorhexidine dihydrocloride caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation nor in the preincubation test each carried out with and without metabolic activation.