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

Repeated dose toxicity: dermal

Administrative data

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

For chlorhexidine digluconate itself there are no standardised studies on dermal toxicity available performed according to recent guidelines. Therefore, based on the following scientific justification data for chlorhexidine diacetate are taken into further consideration. Chlorhexidine digluconate consists of chlorhexidine and gluconate in a molar ratio of 1:2. In aqueous solutions, Chlorhexidine diglucoante will dissociate and the constituents will be present as non-ionic and ionic species, depending on the pH of the solution. As indicated by the pKa values for chlorhexidine and gluconic acid, chlorhexidine digluconate will be forming double protonated chlorhexidine cations and single deprotonated gluconic acid (gluconate) anions in aqueous solutions at biologically relevant pH values. It can be reasonably assumed that other chlorhexidine salts such as the diacetate and the dihydrochloride will also dissociate at biologically relevant pH values. The data on the different chlorohexidine salts show the same toxicological properties, i.e. the compounds have low acute toxicity, are not irritating or slightly irritating to skin, irritating to eyes, not sensitizing, not genotoxic and not teratogenic. Subchronic oral toxicicity studies in rats are reported with chlorhexidinine gluconate and chlorhexidine acetate. The only effects, found for both compounds were reduced body weight gain and the presence of giant cells in the abdominal lymph nodes. The LOAEL, given as concentration of chlorhexidine, was the same for both substances (48.5 mg/kg bw in the study with the gluconate and 47.8 mg/kg bw in the study with the acetate). Based on this information the toxicological properties of the different chlorhexidine salts can be attributed to the chlorhexidine moiety. In regard to this specific endpoint for both substances, chlorhexidine digluconate and chlorhexidine diacetate, the acute dermal toxicity is low (LD50 >2000mg/kg bw and >5000 mg/kg bw, respectively) and also for other endpoints regarding skin (irritation and sensitisation) both compounds do not show adverse properties. The pH values are in a similar range of 5-7. Due to the limited dermal absorption of chlorhexidine digluconate (e.g. in rats >3%), which based on the similar physico-chemical properties should also apply to chlorhexidine diacetate, the overall risk for systemic toxicity induced by dermal exposure should be rather limited and similar for both substances. Additionally, both anions, acetate and gluconate, are not known to induce dermal systemic toxicity. Based on these properties the inclusion of this study for repeated dose dermal toxicity conducted with chlorhexidine diacetate is considered to properly address this endpoint also for chlorhexidine digluconate. This internal company report is not available for public. However, methods and results have been reported in a summarised format by Cal EPA and US EPA.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Information on the read across justification is availablle under Chapter 13.2 Other assessment reports

Cross-reference

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

sub-chronic toxicity: dermal

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Information on the read across justification is availablle under Chapter 13.2 Other assessment reports

Reason / purpose for cross-reference:

read-across source

Species:

rat

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

-Age at study initiation: ca. 12 weeks
No further detailed information available

Type of coverage:

occlusive

Vehicle:

water

Details on exposure:

The test substance was applied moistened with 2 ml of water to the intact dorsal dermal surface and the treatment area was covered with a gauze dressing and wrap.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

13 consecutive weeks

Frequency of treatment:

6 h/d on 5 d/week

Remarks:

Doses / Concentrations:
0, 250, 500 or 1000 mg/kg bw/d

No. of animals per sex per dose:

10 m / 10 f

Control animals:

yes

Details on study design:

The dose selection was justified as being a limit dose of 1000 mg/kg bw/d and choosing logarithmically spaced doses for the mid- and low-dermal doses.
No further detailed information available.

Observations and examinations performed and frequency:

see below

Sacrifice and pathology:

No further detailed information available.

Statistics:

Fishers exact test

Details on results:

Body weight and food consumption were slightly lowered during the initial 2 weeks of the study. At all doses minimal dermal irritation such as erythema, edema, desquamation and fissuring was observed.
There were no adverse effects on ophthalmology or haematology.
In females treated with >= 500 mg/kg bw/d the activities of aspartate aminotransferase and of alanine aminotransferase were increased at the end of the study.
The incidence of hepatocellular degeneration/necrosis in males was 0, 0, 0, 1 and 0, 1, 5, and 7 in females. However, the liver necrosis was graded as minimal in all animals and the incidence of 1/10 at 250 mg/kg bw/d was not statistically significant.

Dose descriptor:

NOEL

Effect level:

360 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: A dermal NOEL was not established as there were local effects at all doses in both sexes. For systemic effects a NOEL of 250 mg/kg bw/d (corresponding to 360 mg chlorhexidine diglucon ate/kg bw/d) was derived based on liver findings at higher doses.

Critical effects observed:

not specified

Conclusions:

Under the conditions of the assay, dermal application of chlorhexidine diacetate to rabbits for 13 weeks led to minimal liver necrosis in animals dosed with >= 500 mg/kg bw/d. A dose of 250 mg/kg bw/d was associated with local effects such as irritation but there were no statistically significant systemic effects.

Executive summary:

For chlorhexidine digluconate itself there are no standardized studies available performed according to recent guidelines. Therefore, data for chlorhexidine diacetate have been taken into further consideration.

In a 13 -week dermal toxicity study with NZW rabbits, chlorhexidine acetate was applied at doses of 0, 250, 500, or 1000 mg/kg bw/d to the intact dorsal dermal surface on 6 h/d and 5 d/week under occlusive conditions.

Body weight and food consumption were slightly lowered during the initial 2 weeks of the study. At all doses minimal dermal irritation such as erythema, edema, desquamation and fissuring was observed. There were no adverse effects on ophthalmology or haematology.

In females treated with >= 500 mg/kg bw/d the activities of aspartate aminotransferase and of alanine aminotransferase were increased at the end of the study.

The incidence of hepatocellular degeneration/necrosis in males was 0, 0, 0, 1 and 0, 1, 5, and 7 in females. However, the liver necrosis was graded as minimal in all animals and the incidence of 1/10 at 250 mg/kg bw/d was not statistically significant.

A dermal NOEL was not established as there were local effects at all doses in both sexes. For systemic effects a NOEL of 250 mg/kg bw/d (corresponding to 360 mg chlorhexidine digluconate/kg bw/d) was derived based on liver findings at higher doses.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1988

Reference 2

Reference Type:

secondary source

Title:

Summary Of Toxicology Data. Chlorhexidine Diacetate

Author:

Cal EPA

Year:

2003

Bibliographic source:

Medical Toxicology Branch. Department Of Pesticide Regulation. California Environmental Protection Agency

Reference 3

Reference Type:

secondary source

Title:

Reregistration Eligibility Decision (RED). Chlorhexidine Diacetate

Author:

US EPA

Year:

1996

Bibliographic source:

Office of Prevention, Pesticides and Toxic Substances (OPPTS). United States Environmental Protection Agency (US EPA), Washington, D.C.

Materials and methods

Principles of method if other than guideline:

No detailed information available

GLP compliance:

not specified

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Chlorhexidine di(acetate)

EC Number:

200-302-4

EC Name:

Chlorhexidine di(acetate)

Cas Number:

56-95-1

Molecular formula:

C22H30Cl2N10.2C2H4O2

IUPAC Name:

N',N'''''-hexane-1,6-diylbis[N-(4-chlorophenyl)(imidodicarbonimidic diamide)] diacetate

Details on test material:

-Purity: 97.7 %
-Lot/batch No.: R4796

Test animals

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

-Age at study initiation: ca. 12 weeks
No further detailed information available

Administration / exposure

Type of coverage:

occlusive

Vehicle:

water

Details on exposure:

The test substance was applied moistened with 2 ml of water to the intact dorsal dermal surface and the treatment area was covered with a gauze dressing and wrap.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

13 consecutive weeks

Frequency of treatment:

6 h/d on 5 d/week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 250, 500 or 1000 mg/kg bw/d
Basis:

No. of animals per sex per dose:

10 m / 10 f

Control animals:

yes

Details on study design:

The dose selection was justified as being a limit dose of 1000 mg/kg bw/d and choosing logarithmically spaced doses for the mid- and low-dermal doses.
No further detailed information available.

Examinations

Observations and examinations performed and frequency:

see below

Sacrifice and pathology:

No further detailed information available.

Statistics:

Fishers exact test

Results and discussion

Results of examinations

Details on results:

Body weight and food consumption were slightly lowered during the initial 2 weeks of the study. At all doses minimal dermal irritation such as erythema, edema, desquamation and fissuring was observed. There were no adverse effects on ophthalmology or haematology.
In females treated with >= 500 mg/kg bw/d the activities of aspartate aminotransferase and of alanine aminotransferase were increased at the end of the study.
The incidence of hepatocellular degeneration/necrosis in males was 0, 0, 0, 1 and 0, 1, 5, and 7 in females. However, the liver necrosis was graded as minimal in all animals and the incidence of 1/10 at 250 mg/kg bw/d was not statistically significant.

Effect levels

Dose descriptor:

NOEL

Effect level:

360 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: A dermal NOEL was not established as there were local effects at all doses in both sexes. For systemic effects a NOEL of 250 mg/kg bw/d (corresponding to 360 mg chlorhexidine digluconate/kg bw/d) was derived based on liver findings at higher doses.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of the assay, dermal application of chlorhexidine diacetate to rabbits for 13 weeks led to minimal liver necrosis in animals dosed with >= 500 mg/kg bw/d. A dose of 250 mg/kg bw/d was associated with local effects such as irritation but there were no statistically significant systemic effects.

Executive summary:

For chlorhexidine digluconate itself there are no standardized studies available performed according to recent guidelines. Therefore, data for chlorhexidine diacetate have been taken into further consideration.

In a 13 -week dermal toxicity study with NZW rabbits, chlorhexidine acetate was applied at doses of 0, 250, 500, or 1000 mg/kg bw/d to the intact dorsal dermal surface on 6 h/d and 5 d/week under occlusive conditions.

Body weight and food consumption were slightly lowered during the initial 2 weeks of the study. At all doses minimal dermal irritation such as erythema, edema, desquamation and fissuring was observed. There were no adverse effects on ophthalmology or haematology.

In females treated with >= 500 mg/kg bw/d the activities of aspartate aminotransferase and of alanine aminotransferase were increased at the end of the study.

The incidence of hepatocellular degeneration/necrosis in males was 0, 0, 0, 1 and 0, 1, 5, and 7 in females. However, the liver necrosis was graded as minimal in all animals and the incidence of 1/10 at 250 mg/kg bw/d was not statistically significant.

A dermal NOEL was not established as there were local effects at all doses in both sexes. For systemic effects a NOEL of 250 mg/kg bw/d (corresponding to 360 mg chlorhexidine digluconate/kg bw/d) was derived based on liver findings at higher doses.