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

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1978 to 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The results of the pharmacokinetic part of the study are available in summary only, the detailed results are presented in an additional part of the report which has not been available for evaluation. This is not considered to be of major significance for the evaluation of the toxicity part of the study which was performed comparable to OECD guideline 452 (1981). The findings are considered relevant and the present documentation is considered sufficient to evaluate the chronic toxicity of chlorhexidine digluconate.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1982

Report date:

1982

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

GLP compliance:

yes

Remarks:

Auditing in accordance with ICI’s policies and procedures for GLP

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

242-354-0

EC Name:

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

Cas Number:

18472-51-0

Molecular formula:

C22H30Cl2N10.2C6H12O7

IUPAC Name:

N',N'''''-hexane-1,6-diylbis[N-(4-chlorophenyl)(imidodicarbonimidic diamide)] - D-gluconic acid (1:2)

Test animals

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: ICI, Alderley Park Breeding Unit, UK
Age/weight at study initiation: 11-19 months / 9.7-16.8 kg

Administration / exposure

Route of administration:

other: orally via gelatine capsules

Vehicle:

water

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

6 and 12 months

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 0.5, 5 and 40/25 mg/kg bw/d (given as chlorhexidine base; highest dose was reduced after 28 w)
Basis:

No. of animals per sex per dose:

6 months: 3 m /3 f
12 months: 5 m / 5 f
12 months + 2 months recovery (only controls and high-dose group): 3 m / 3 f

Control animals:

yes

Details on study design:

Group 1 (Dose (mg/kg bw) / Concentration (%) / Applied volume (mL/d)): 0 / 0 / 0
Group 2 (Dose (mg/kg bw) / Concentration (%) / Applied volume (mL/d)): 0.5 / 1 / 1-1.4
Group 3 (Dose (mg/kg bw) / Concentration (%) / Applied volume (mL/d)): 5 / 10 / 1-1.4
Group 4 (Dose (mg/kg bw) / Concentration (%) / Applied volume (mL/d)): 40/25* / 10 / 7-12 (5-8)

Dose given as chlorhexidine base; * Dose reduction after 28 weeks

Post-exposure period: 2 months (controls and high-dose group dosed over 12 months)

Examinations

Observations and examinations performed and frequency:

Clinical signs: daily
Mortality: Yes
Body weight: weekly
Food consumption: daily
Water consumption: No
Ophthalmoscopic examination: Yes, at pre-dose, at 1, 3, 6, 9 and 12 months, and terminally for dogs used for withdrawal studies (from group I and IV)
Haematology: Yes, all animals, time points: pre-dose, 1, 3, 6, 9, 12 months, and in withdrawal studies (group I and IV) at termination of withdrawal period
Clinical Chemisty: Yes, all animals, time points: pre-dose, 1, 3, 6, 9, 11, 12 months, and in withdrawal studies (group I and IV) 1 and 2 months after dosing had ceased
Urinalysis: Yes, number of animals: group I and IV: 5 m/5 f scheduled to be killed after 12 months, group I and IV: 3 m/3 f used for withdrawal studies, time points: pre-dose, 1, 3, 6, 9, 12 months

Sacrifice and pathology:

Organ Weights: Yes
Gross and histopathology: Yes, all dose groups and all major organs

Other examinations:

Toxicokinetics: Chlorhexidine and p-chloroaniline concentrations in organs
Number of animals: all animals
Blood: pre-dose, 1, 3, 6, 9, 12 months, and in withdrawal studies (group I and IV) at termination of withdrawal period
Organs (brain, kidney, liver, lung, axillary lymph node and a lymph node of the mesenteric chain) at necropsy

Statistics:

Analysis of variance; Student’s t-test

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Details on results:

Clinical signs:
Group IV: Treatment-related findings were vomiting, reluctance to accept dosing, and alopecia and staining of hair. The incidence of vomiting decreased after the dose was reduced from 40 to 25 mg/kg bw/d; Group II and III: Sporadic vomiting occurred on altogether seven occasions compared to one in group I (control). No other treatment-related effects in these groups.

Mortality:
Group IV: Two dogs were killed on humane reasons due to intercurrent disease, one after 80 days, the other after 3.5 months. Histopathology revealed bronchopneumonia. It could not be excluded that dosing with chlorhexidine digluconate exacerbated the disease and was an indirect contributory factor in the cause of death. No treatment-related effects in other groups.

Body weight gain:
Group IV: Loss of body weight during the first 6 months during treatment with 40 mg/kg bw/d, gradual increase of body weight afterwards when the dose was reduced to 25 mg/kg bw/d. Initial body weight regained after 12 months. No treatment-related effects in other groups.

Food consumption and compound intake:
Group IV: Reduced during the first 6 months (at a dose of 40 mg/kg bw/d), by about 10 – 40 %, in females more than in males; less pronounced reduction at 25 mg/kg bw/d.

Clinical chemistry:
Group IV: Significantly increased levels of alanine aminotransferase (ALT) from the 3rd month on and – less pronounced and not at all time points – of aspartate aminotransferase; significantly lower levels of total protein and albumin. The elevated ALT decreased after dose reduction. Other findings in group IV and sporadically in group II and III were lower total bilirubin, blood sugar, sodium and calcium and higher levels of urea at various time points during the course of study. However, these findings did not show a consistent temporary pattern and showed no dose-related trend. They are therefore not considered to be related to treatment.

Urinalysis:
Group IV: Increased specific gravity of urine (all time points except 9 months) and interim (3 and 6 months) significantly lowered pH. These effects are considered to be treatment-related. No treatment-related effects in the other groups.

Gross and histopathology:
Group III and IV: At terminal sacrifice, there was an increase in macroscopic observations for the liver. The observed changes were white capsular striations and/or dark areas and, in one dog in group IV, a mottled, yellow, firm and shrunken liver. Histopathology revealed treatment-related hepatic changes after 6 and after 12 months and after the 2-month recovery period. In animals of group IV, changes were characterised by focal degeneration, necrosis and loss of hepatocytes and, in a few dogs, extensive irregular areas of necrosis and loss of hepatocytes over several liver lobules. Changes associated with these findings were focal mononuclear infiltrations, haemorrhages, bile duct hyperplasia, fibrosis, haemosiderosis, and acute inflammatory cell infiltrates. In animals of group III, treatment-related changes were characterised by centrilobular fibrosis. Other treatment-related findings in animals of group IV were observed in the stomach. These were characterised by slight to minimal haemorrhages in the subepithelial lamina propria. These effects are probably associated with the increased incidence of vomiting. No other treatment-related effects were noted in animals of group IV and III. There were no treatment-related effects in animals of group II.

Except for some spurious positives, no measurable concentrations of chlorhexidine or p-chloroaniline were found in blood of group I (control), group II, and group III animals. The concentration of chlorhexidine in organs and blood increased with the administered dose. The highest concentrations were found in the liver, followed by kidney, mesenteric lymph nodes, lung, axillary lymph nodes, brain and blood. The concentrations in liver, kidney and lung were similar after 6 and 12 months indicating the achievement of steady state in the corresponding organ. Data of the withdrawal part of the study indicate that clearance from the tissues was slow.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

0.5 other: mg/kg bw/day (given as chlorhexidine base)

Sex:

male/female

Basis for effect level:

other: Equivalent to 0.89 mg/kg bw/day as chlorhexidine digluconate

2

Dose descriptor:

LOAEL

Effect level:

5 other: mg/kg bw/day (given as chlorhexidine base)

Sex:

male/female

Basis for effect level:

other: Hepatic centrilobular fibrosis Equivalent to 8.88 mg/kg bw/day as chlorhexidine digluconate

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

NO(A)EL: 0.5 mg chlorhexidine base/kg bw/d (equivalent to 0.89 chlorhexidine digluconate/kg bw/d)
LO(A)EL: 5 mg chlorhexidine base/kg bw/d (equivalent to 8.88 chlorhexidine digluconate/kg bw/d) (based on hepatic centrilobular fibrosis)

Executive summary:

Chlorhexidine digluconate was administered orally in capsules to male and female dogs for up to one year. A high-dose recovery groups was included to investigate potential reversibility of effects. The dosage regimen was 0, 0.5, 5 and 40/25 mg/kg bw/d (given as as chlorhexidine base; highest dose was reduced after 28 w).

Under the conditions of the study, chlorhexidine digluconate caused a decrease in body weight, increased vomiting, reluctance to accept dosing, and alopecia and staining of hair in the high-dose group (group IV). The incidence of vomiting decreased after the dose was reduced from 40 to 25 mg/kg bw/d. In the high-dose group, two animals were killed for humane reasons because of bad conditions. These animals suffered from bronchopneumonia and it could not be excluded that dosing with chlorhexidine digluconate exacerbated the disease and was an indirect contributory factor in the cause of death. At the high-dose group, clinical chemistry showed increased levels of alanine aminotransferase and of aspartate aminotransferase and significantly lower levels of total protein and albumin. These findings indicated effects on the liver. Gross and histopathology also revealed that the liver was a target organ. Changes were characterised by focal degeneration, necrosis and loss of hepatocytes; associated findings were monuclear infiltrations, haemorrhages, bile duct hyperplasia, fibrosis, haemosiderosis, and acute inflammatory cell infiltrates. In animals of group III, treatment-related changes were characterised by hepatic centrilobular fibrosis. Other organs were not affected except for a probable relationship between slight to minimal haemorrhages in the stomach and the incidence of vomiting which in turn was related to chlorhexidine digluconate treatment. No effects on the liver were observed at the lowest dose (group II).

NO(A)EL: 0.5 mg chlorhexidine base/kg bw/d (equivalent to 0.89 chlorhexidine digluconate/kg bw/d)

LO(A)EL: 5 mg chlorhexidine base/kg bw/d (equivalent to 8.88 chlorhexidine digluconate/kg bw/d) (based on hepatic centrilobular fibrosis)