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

Carcinogenicity

Description of key information

Carcinogenicity studies were performed with oral administration of chlorhexidine to rats and mice. In both studies there was no evidence for a carcinogenic activity.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1978-03-02 to 1980-05-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

not applicable

GLP compliance:

yes

Remarks:

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

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: ICI, Alderley Park Breeding Unit, UK
- Age at study initiation: 3-5 weeks
- Weight at study initiation: 46-128 g (m); 41-120 g (f)
- Housing: RCl cages, 5 of one sex in each cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2
- Humidity (%): 50 +/- 15
- Air changes (per hr): >15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: feed

Details on exposure:

Rate of preparation of diet (frequency): Dietary concentrations were adjusted weekly until week 58 and then fortnightly.
Vehicle: Chlorhexidine digluconate was added to the diet as a 20 % aqueous solution (equivalent to 11.26 % chlorhexidine base).
The concentration of chlorhexidine in the diet was adjusted weekly and routinely monitored throughout the study and was between 95 % and 105 % of the theoretical value except for a few values.
Three similarly constituted groups of rats received unmedicated diet and served to generate contemporaneous control data. Additional groups of 50 male and 50 female rats also received the test substance at the appropriate dosage, or unmedicated diet and were assigned to interim sacrifice after 4, 13, 26, 39 52, 65, 78, 91 weeks of treatment. With the exception of weeks 4 and 13 when 10 rats per sex from each group were sacrificed, 5 rats per sex per group were sacrificed after each elapsed time period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity: by blending in a srew type mixer

Duration of treatment / exposure:

105 weeks

Frequency of treatment:

continuously via diet

Post exposure period:

none

Remarks:

Doses / Concentrations:
0, 5, 25 or 50 mg/kg bw/d (as chlorhexidine base); ca. 0, 8.9, 44.5 or 89 mg/kg bw/d (as chlorhexidine digluconate)
Basis:
nominal in diet

No. of animals per sex per dose:

50 m / 50 f

Control animals:

yes

Details on study design:

Interim sacrifice(s): after 4, 13, 26, 39, 52, 65, 78 and 91 weeks

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
-Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS:
-Time schedule: once weekly

BODY WEIGHT:
-Time schedule for examinations: on day 0 and weekly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE:
-Weight of food consumed by each cage of mice was calculated weekly (food offered / food remaining)

FOOD EFFICIENCY:
-Group mean food conversion ratios were calculated at weekly intervals for the first 13 weeks and at quarterly intervals thereafter

WATER CONSUMPTION:
-Time schedule for examinations: once daily

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

Sacrifice and pathology:

Final sacrifice: after 105 weeks
Histopathology: At termination of study from all dose groups with all major organs

Other examinations:

The concentration of chlorhexidine and of p-chloroaniline in the blood and organs were assessed at all interim sacrifice time points and at the end of the study.

Statistics:

yes

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Body weight/Weigth gain:
50 mg/kg bw/d (males) and >= 25 mg/kg bw/d (females): decreased compared with respective control from week 39 of treatment

Macroscopic investigations:
About 30 % of all animals had lesions of the hard palate after 45-46 weeks. The appearance of this lesion was not related to the administration of chlorhexidine digluconate but to the presence of the fibres in the non-pelleted diet. The incidence of animals bearing palpable tumours and the total numbers of palpable swellings were not increased by treatment with chlorhexidine digluconate.

Histopathology:
No increased incidences of benign and malignant neoplasms of individual tumour types. In rats from all groups, periodontal ulcers and sinuses with inflammation were seen. These were caused by long, sharp vegetable fibres in the diet that was fed to the animals during the initial 45 weeks. Squamous cell carcinomas, mostly on the base of chronically inflamed ulcers and sinuses, developed in a total number of 74 animals from all groups. There was, however, no relationship between the development of the described hard palate lesions or these tumours and the presence of chlorhexidine digluconate in the diet. The only observation related to administration of chlorhexidine digluconate was the increase in the percentage number of animals showing the presence of pigment-laden macrophages in the mesenteric lymph nodes (severity grade II).

Other examinations:
The concentration of chlorhexidine in blood, brain, lung, liver, kidney and mesenteric lymph nodes increased with dose of chlorhexidine digluconate indicating that at least some of the test substance was absorbed from diet. The concentration of chlorhexidine in the brain was consistently lower than that in the blood, while the concentrations in blood, liver and lung were comparable. The highest concentration was found in the kidneys and in the mesenteric lymph nodes after 21 months of feeding chlorhexidine digluconate. The concentrations of p-chloroaniline in the mentioned organs and in blood were very low throughout the study and at the limit of detection at many time points.

Time to tumours:
The times of appearance of palpable swellings was not different between groups of animals treated with chlorhexidine digluconate and controls.

Dose descriptor:

LOEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: pigment-laden macrophages in mesenteric lymph nodes (grade II)

Conclusions:

Under the conditions of the present study, there was no evidence for a carcinogenic effect of chlorhexidine digluconate in rats following oral administration of doses up to the MTD.

Executive summary:

Carcinogenicity study with dietary administration of test compound to rats for at least 105 weeks.

Body weight and body weight gain in male rats at 50 mg/kg bw/d and in female rats at >= 25 mg/kg bw/d were slightly lower than that of the corresponding control groups indicating that the maximum tolerated dose (MTD) was reached.

Kinetic data show that at least some of the chlorhexidine is absorbed from the diet. The concentration of chlorhexidine found in blood and various organs of animals increased with dose and time. The highest concentrations were found in the kidneys and in the mesenteric lymph nodes.

There was no evidence of a chlorhexidine-related increase in the incidence of any benign or malignant tumour. Neither the distribution among the groups nor the predominant macropathological entities identified at necropsy showed any effects that could be related to the treatment of the animals with chlorhexidine digluconate. The only histological effect that is attributed to the treatment with chlorhexidine digluconate is an increase in the percentage number of animals showing the presence of pigment-laden macrophages in the mesenteric lymph nodes.

LOEL (based on pigment-laden macrophages in mesenteric lymph nodes, grade II): 5 mg chlorhexidine mg/kg bw/d (equal to 8.9 mg chlorhexidine digluconate/kg bw/d).

The treatment gave no evidence for a carcinogenic effect of chlorhexidine digluconate.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LOAEL

8.9 mg/kg bw/day

Study duration:

chronic

Species:

rat

Justification for classification or non-classification

Carcinogenicity studies were performed with oral administration of chlorhexidine to rats and mice. In both studies there was no evidence for a carcinogenic activity.

Therefore, there is no need for a classification.

Additional information

The results of carcinogenicity studies in animals are summarised in the following Table:

Route	Species Strain Sex no/group	dose levels frequency of application	Tumours
Oral, food	Mouse C57 BL/10J male and female 52 animals/group/sex	0, 100, 200, 400 mg chlorhexidine/kg bw d = 0, 175, 350, 700 or 1400 mg chlorhexidine digluconate/kg bw d; daily	The incidence and distribution of benign and malignant tumours showed no substance-related changes
Oral, food	Rat Wistar derived male and female 50 animals/group/sex	0, 5, 25, 50 mg chlorhexidine/kg bw d = 0, 8.8, 44 or 88 mg Chlorhexidine digluconate/kg bw d; daily	The incidence and distribution of benign and malignant tumours showed no substance-related changes