D-gluconic acid, compound with N,N''-bis(4-chlorophenyl)-3,12-diimino-2,4,11,13-tetraazatetradecanediamidine (2:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

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:

2001-12-12 to 2002-03-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

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, 0.1, 0.316, 1, 3.16 and 10 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 >= 10 µg/plate.

Vehicle / solvent:

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

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: without metabolic activation: sodium azide (TA 1535, TA 100); 2-nitrofluorene (TA 98); 9-aminoacridine (TA 1537); methylmethanesulfonate (TA 102); with metabolic activation: cyclophosphamide (TA 100, TA 1535); 2-anthraceneamide (TA 98, TA 102, TA 1537)

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

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

plate incorporation test: without S9 mix: at 10 ug/plate; with S9 mix: no cytotoxicity; preincubation test: without S9 mix: all strains at >= 3.16 ug/plate; with S9 mix: at 10 ug/plate

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

plate incorporation test: without S9 mix: at 10 ug/plate; with S9 mix: no cytotoxicity; preincubation test: without S9 mix: >= 3.16 ug/plate

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

plate incorporation test: without S9 mix: at 10 ug/plate; with S9 mix: no cytotoxicity; preincubation test: without S9 mix: >= 3.16 ug/plate; with S9 mix: at 10 ug/plate

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

plate incorporation test: without S9 mix: at 10 ug/plate; with S9 mix: no cytotoxicity; preincubation test: without S9 mix: >= 3.16 ug/plate; with S9 mix: at 10 ug/plate

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

plate incorporation test: without S9 mix: at >= 3.16 ug/plate; with S9 mix: no cytotoxicity; preincubation test: without S9 mix: at >= 3.16 ug/plate; with S9 mix: at 10 ug/plate

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results: negative

No evidence for mutagenic activity in the Salmonella typhimurium assay.

Executive summary:

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. 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 >= 10 µg/plate. Under the conditions of the assay where positive controls exerted the expected mutagenic effects, chlorhexidine digluconate 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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

No explicit positive control was run with the test. However, several of the different test substances gave clearly positive results in the absence and presence of an exogenous metabolic activation indicating the validity of the assay under the performed conditions. The number of metaphases scored was only 100. However, there was a clear lack of a cytogenetic effect of chlorhexidine digluconate although the cells were continuously treated for a time equivalent of 1.5 normal cell cycle lengths. Therefore, the study is considered as comparable with the corresponding OECD guideline and the results of the study are considered relevant for the assessment of in vitro cytogenicity of chlorhexidine digluconate.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

see above

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: Syrian hamster embryo cells (SHE)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (rat, liver, induced with sodium phenobarbital and benzoflavone)

Test concentrations with justification for top dose:

0, 0.3, 1, 3 and 10 µM

Vehicle / solvent:

The final test concentrations were obtained by first diluting with deionized water to 10 mg/mL and further with culture medium to the desired concentrations.

Negative solvent / vehicle controls:

yes

Positive controls:

other: No explicit positive control was run with the test. However, several of the different test substances gave clearly positive results indicating the validity of the assay under the performed conditions

Details on test system and experimental conditions:

Tertiary cultures of SHE cells were treated with the test chemical for 24 h, i.e. 1.5 times the doubling time of the cells (16 h). In the presence of exogenous metabolic activation system, cells were treated with the test substance for 3 h followed by 18 h incubation in fresh medium. Cultures were treated 3 h before harvest with colcemide. Cytotoxicity was determined as colony-forming efficiency (relative to control) relative after 24 h of treatment with test substance and subsequent incubation for 7 days for colony formation.

Evaluation criteria:

For determination of chromosome aberrations, 100 metaphases were scored per experimental group. Achromatic lesions greater than the width of the chromatid were scored as gaps unless there was displacement of the broken piece of chromatid. If there was displacement, they were scored as breaks.

Species / strain:

other: Syrian hamster embryo cells (SHE)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

10 µM (-S9 mix)

Vehicle controls validity:

valid

Conclusions:

Interpretation of results: negative
No induction of chromosomal aberrations in a cytogenicity assay with SHE cells in the presence or absence of metabolic activation.

Executive summary:

Cytogenicity study in mammalian cells (Syrian hamster embryo cells, SHE) comparable to OECD guideline 473 with and without metabolic activation by S9 mix from phenobarbital/benzoflavone-induced rat liver. The test substance was applied up to the cytotoxic concentration range. Under the conditions of the assay where a number of other, simultaneously tested substances gave a positive response, chlorhexidine digluconate tested up to cytotoxic concentration of 10 µM did not induce chromosomal aberrations in a cytogenicity assay with SHE cells in the presence or absence of metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001-12-12 to 2002-06-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (Aroclor 1254-induced rat liver)

Test concentrations with justification for top dose:

1st trial: 0, 0.1, 0.5, 1, 5 and 10 ug/ml; 2nd trial: 1st trial: 0, 0.5, 1, 5, 10 and 20 µg/mL.
The top concentrations were chosen based on a preliminary cytotoxicity experiment, where pronounced cytotoxicity was observed at >= 10 µg/mL (+/- S9 mix).

Vehicle / solvent:

The final test concentrations were obtained by dilution with culture medium which was then added to the cultures.

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: -S9 mix: ethylmethanesulphonate; +S9 mix: 9,10-dimethylbenzanthracene

Details on test system and experimental conditions:

Duration of exposure: 24 hrs without S9 mix and 4 hrs with S9 mix

Evaluation criteria:

If the test substance in both experiments does not increase the mutation frequency 2-fold above the mean of the solvent controls under any condition, or if the mutation frequency is always < 20 * 10E-6 and if at least 1,000,000 cells per condition have been evaluated, the compound is considered as negative in the test.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

+/- S9 mix at >= 10 µg/mL

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results: negative

No evidence for mutagenic activity with and without metabolic activation.

Executive summary:

Mutagenicity study in mammalian cells (V79) according to OECD guideline 476 with and without metabolic activation by S9 mix from Arochlor 1254-induced rat liver. The concentration of the test substance was based on the observations of a pretest in which cytotoxicity was observed from >= 10 µg/mL. Under the conditions of the assay where positive controls exerted the expected mutagenic effects, chlorhexidine digluconate tested up to cytotoxic concentrations of 10 or 20 µg/mL did not induce forward mutations in the HPRT-V79 -mutagenicity assay in the presence or absence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

No official statement concerning GLP status, but study was controlled and signed by responsible study director. Only male mice were used. Cells from bone marrow were collected only once, at 6 h after the final treatment. Although there was no mortality at any dose, results were only reported for 6/10 treatment group. However, the presented results were consistent and clearly negative throughout. The positive control gave a positive response indicating the principle validity of the assay conditions. In summary, the results of the study are considered relevant as supportive data for the assessment of the genotoxicity of chlorhexidine digluconate.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

no

Remarks:

No official statement concerning GLP status, but study was controlled and signed by responsible study director

Type of assay:

micronucleus assay

Species:

mouse

Strain:

Swiss

Sex:

male

Details on test animals or test system and environmental conditions:

Source: C.N.R.S, Orleans la Source
Age at study initiation: no data
Weight at study initiation: 30 g

Route of administration:

intraperitoneal

Vehicle:

Vehicle: DMSO/glycerol (1+4 v/v)
Concentration in vehicle: no data

Details on exposure:

Total volume applied: 10 or 20 mL/kg bw

Duration of treatment / exposure:

Number of applications: 2
Interval between applications: 24 h

Post exposure period:

6 h

Remarks:

Doses / Concentrations:
2 * 10, 2 * 20 or 2 * 30 mg/kg bw
Basis:

No. of animals per sex per dose:

at least 5 m per dose and sampling time

Control animals:

yes, concurrent vehicle

Positive control(s):

Substance used as Positive Control: 2 * 2 mg/kg bw Mitomycin C (vehicle: water)

Tissues and cell types examined:

Tissue: bone marrow

Details of tissue and slide preparation:

Maximum tolerable dose: 30 mg/kg bw
Number of animals: all animals
Number of cells: 2000
Time points: 6 h after last treatment
Type of cells: erythrocytes in bone marrow

Evaluation criteria:

Parameters: numbers and types of structural aberrations and ratio polychromatic/normochromatic erythrocytes

Statistics:

not further specified

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

pilorection, dyspnoea, ataxie, pain at injection site

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results: negative

No increase of micronuclei in bone marrow cells at any dose above vehicle control. The positive control (Mitomycin) gave the expected positive response.

Executive summary:

Micronucleus test with male Swiss mice with intraperitoneal administration of chlorhexidine digluconate using three different dose levels and two treatments at 24 h interval. Assessment of micronuclei in bone marrow 6 h after the second treatment. Under the conditions of the assay, chlorhexidine digluconatedid not cause an increase in the frequency of micronuclei in bone marrow cells of male mice.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

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

Genotoxicity in vitro of 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

Chlorhexidine digluconate 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 digluconate did not reveal any cytogenetic activity (induction of chromosomal aberrations) when tested up to cytotoxic concentrations. 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 digluconate 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 digluconate did not induce sister chromatid exchange (SCE) in SHE cells (tested only in the absence of exogenous metabolic activation) in a further investigation of the same study group.

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

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

 

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 digluconate have been tested in several in vitro (e.g. Ames test, Chromosomal aberrations, HPRT test) and in vivo systems (Micronucleus test) and in all reliable and relevant tests negative results were obtained.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

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