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Additional information

The results of in vitro genotoxicity studies with chlorhexidine (using chlorhexidine digluconate salt) in animals are summarised in the following Table.

Genotoxicity in vitro of chlorhexidine base tested as chlorhexidine digluconate

Test system
Method
Guideline

Organism/
strain(s)

Concentrations tested*

Result

Remark

- S9

+ S9

Ames test, reverse mutation in prokaryotes, OECD guideline 471

Salmonella typhimuriumTA98, TA100, TA102, TA1535, TA1537

Plate incorporation assay: 0.1-10 µgchlorhexidine digluconate/plate

Preincubation assay: 0.1-10 µgchlorhexidine digluconate/plate

-

-a

Guideline study

Cytotoxicity:

Plate incorporation assay at 3.16 µg/plate (-S9)

Preincubation assay: at 3.16 µg/plate (‑ S9), at 10 µg/plate (+S9)

Chromosomal aberrations, in-house method

Syrian hamster embryo cells (SHE)

0.3-10 µM

-

-b

Comparable to guideline study

Cytotoxicity at 10 µM (‑S9)

Sister chromatid exchange, in-house method

Syrian hamster embryo cells (SHE)

0.3-10 µM

-

n.p.c

Cytotoxicity at 10 µM

HPRT test, forward point mutation in mammalian cells, OECD guideline 476

V79 Chinese hamster lung fibroblasts

0.1-20 µ/mlchlorhexidine digluconate

-

-

Guideline study

Cytotoxicity at 10 µg/ml (+ and ‑S9)

a: Activation system S9 fraction from Arochlor 1254-induced rat liver;
b: Activation system S9 fraction from Phenobarbital/benzoflavone-induced rat liver
c: not performed

* Concentrations tested in terms of chlorhexidine digluconate resulting in the following dose levels for chlorhexidine base:

Ames test: 0.05 - 5.63 µM

Chromosomal aberration: 0.17 - 5.63 µM

Sister chromatid exchange: 0.17 - 5.63 µM

HPRT: 0.05 - 11.26 µM

Chlorhexidine when tested as chlorhexidine digluconate salt was not mutagenic in a Salmonella typhimurium preincubation and a plate incorporation assay performed according to OECD guideline 471.

In a cytogenetic study with Syrian hamster embryo (SHE) cells in vitro,chlorhexidine did not reveal any cytogenetic activity (induction of chromosomal aberrations) when tested up to cytotoxic concentrations as chlorhexidine digluconate. There was no explicit positive control substance in the test, but several of the different substances tested in the same investigation gave clearly positive results in the absence and presence of exogenous metabolic activation indicating the validity of the assay under the performed conditions. The number of metaphases scored was lower than recommended in the OECD guideline 473. However, chlorhexidine clearly lacked a cytogenetic activity even though the cells were continuously treated for a time equivalent of 1.5 normal cell cycle lengths. It is concluded that the study is valid and comparable with the corresponding OECD guideline.

Additionally, chlorhexidine did not induce sister chromatid exchange (SCE) in SHE cells (tested only in the absence of exogenous metabolic activation using the chlorhexidine digluconate salt) in a further investigation of the same study group.

In a HPRT-mutagenicity study in vitro according to OECD guideline 474, chlorhexidine did not induce forward mutations when tested up to cytotoxic concentrations (also tested as chlorhexidine digluconate salt).

The results of in vivo genotoxicity studies in animals are summarised in the following Table.

Genotoxicity in vivo

Type of test
Method/
Guideline

Species
Strain
Sex
no/group

Frequency of application

Sampling times

Dose levels*

Results

Micro­nucleus test

Mouse, Swiss

 5 males/group

2 times, intra­peritoneal, with 24 h interval

6 h after last treatment

2 x 10, 20, or 30 mg/kg in DMSO/ glycerol

 

Result at all dose levels: -

No indication of a clastogenic effect in the bone marrow

 * Dose levels expressed as chlorhexidine digluconate resulting in 2 x 5.63, 11.26 and 16.89 mg/kg as chlorhexidine base.

A micronucleus test was performed with male mice. The study provided no evidence of a clastogenic effect of chlorhexidine in the bone marrow.


Short description of key information:
Possible genotoxic/mutagenic effects of chlorhexidine have been tested using the chlorhexidine digluconate salt in several in vitro (e.g. Ames test, Chromosomal aberrations, HPRT test) and in vivo systems (Micronucleus test) and in all tests negative results were obtained.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The results of several in vitro and in vivo studies gave no indications for a possible genotoxic/mutagenic effect of chlorhexidine when tested as chlorhexidine digluconate. Therefore, there is no need for a classification.