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Repeated dose toxicity: oral

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Administrative data

Endpoint:
chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1972 to 1975
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The concentration of the test substance in the drinking water not explicitly reported, however, the nominal and the actual mean administered dose levels are reported. No details about the statistical evaluations are reported. The reduced water intake due to the bad palatability of the drinking water at higher doses (50 mg/kg bw in groups IV and V) led to a decreased weight gain of more than 15 % indicating that the maximum tolerated dose was exceeded. The outbreak of respiratory infections caused high mortality during the study and the effect of infection treatment with oxytetracycline is not known. In summary, these limitations are not considered to be of major significance. The study 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 base. The salt chlorhexidine digluconate was used instead of chlorhexidine base. Due to the high level of structural similarity it is considered that the derived data are relevant for the assessment of Chlorhexidine base.In addition, Chlorhexidine digluconate dissociates in aqueous solution to the chlorhexidine base and the gluconate.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1975
Report date:
1975

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
GLP compliance:
no
Remarks:
GLP was not compulsory at the time the study was performed (study was carried out under the supervision of ICI’s pharmaceutical division)
Limit test:
no

Test material

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

Test animals

Species:
rat
Strain:
Wistar
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: Supplied when 5 weeks old, but age at study initiation not reported. Males: 168-170.8 g; Females: 151.9-152.9 g

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
Dosage regimen:

The levels of chlorhexidine (reported as chlorhexidine base) to be administered were calculated on the basis of an estimated daily water intake of 20 ml/rat and was gradually increased at higher doses to overcome the reluctance of the animals to accept the altered palatability at higher concentrations.
Doses were maintained by adjusting the concentrations with each 50 g elevation (first six months) or 25 g elevation (later on) in body weight.
Group V received a chlorhexidine digluconate solution that also contained 500 ppm 4-chloroaniline, a chemical degradation product of chlorhexidine.

Nominal body doses of chlorhexidine base:
Group I (control): 0 mg/kg bw
Group II: 5 mg/kg bw
Group III: 25 mg/kg bw (week 1: 10 mg/kg bw, week 2: 20 mg/kg bw)
Group IV: week 1: 10 mg/kg bw, week 2: 20 mg/kg bw, week 3: 35 mg/kg bw, thereafter 50 mg/kg bw
Group V: week 1: 10 mg/kg bw, week 2: 20 mg/kg bw, week 3: 35 mg/kg bw, thereafter 50 mg/kg bw (+ 0.125 mg/kg bw 4-chloroaniline).
Due to the altered palatability and reduced water consumption in group IV and V, the actual dose was approximately 40 mg/kg bw.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
2 years
Frequency of treatment:
continuously
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 5, 25 and 50 mg/kg bw/d (see above)
Basis:

No. of animals per sex per dose:
Control group: 128 m / 128 f
Each treated group: 112 m /112 f
5 m and 5 f per group were killed at 3, 6, 9, 12 and 18 months for interim analysis
5 m and 5 f per group maintained for recovery observations after the exposure period without further treatment for 3-9 weeks
Control animals:
yes
Details on study design:
Post-exposure period: Recovery group animals were observed 3-9 weeks post exposure

Examinations

Observations and examinations performed and frequency:
Clinical signs: Yes
Mortality: Yes
Body weight: Yes
Food consumption: Yes
Water consumption: Yes
Ophthalmoscopic examination: Yes
Haematology: Yes
Clinical Chemistry: Yes
Urinalysis: Yes
Sacrifice and pathology:
Organ Weights: Yes
Gross and histopathology: Yes, all major organs
Other examinations:
Toxicokinetics: Chlorhexidine concentration in organs/tissue: Blood, brain, lung, liver, kidney, mesenteric lymph node, internal iliac lymph node, blood; Time Points: 3, 6, 12, 18, 21, 24 months and 3, 6, 9 weeks after cessation of treatment
Statistics:
No details reported, no statistical evaluation of histopathology findings

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):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
Clinical signs:
A dehydration condition with alterations in behaviour, piloerection, and weight loss was induced in the 50 mg/kg bw dose groups IV and V as the water intake was reduced, probably due to the reduced palatability of drinking water at higher concentrations of chlorhexidine. This was most evident during the first 3 months. No treatment-related effects were observed in the 5 and 25 mg/kg bw dose groups (II and III).

Mortality:
Mortalities occurred throughout the study in both the control and the treated groups. There were no marked differences in premature deaths apparent between groups, although mortalities seemed to have been slightly higher among animals receiving the higher concentrations (a statistical analysis was not presented).

Body weight gain:
The mean starting weight in the 25 and 50 mg/kg bw groups (III, IV, and V) was significantly but marginally (< 3 g) higher than in the control and the 5 mg/kg bw group. This is attributable to the protracted time period during which the animals were introduced into the experiment. Starting from week 4 and 5, a treatment-related decrease in weight gain was observed at >= 25 mg/kg bw. Weight reduction was more marked in males than in females.

Water intake:
Compared to control, the amount of water intake was significantly lower in rats dosed with >= 25 mg/kg bw throughout the study. This led to a lower than originally calculated intake of chlorhexidine in these groups.

Haematology:
Initially rapid increase in packed cell volume at 50 mg/kg bw (groups IV and V). No treatment-related effect on haemoglobin content, packed cell volume, MCHC, leucocytes, platelets, methaemoglobin, haemopoiesis.

Urinalysis:
Up to 12 months, the volume of urine excreted from treated female and, more pronounced, male rats was smaller than that from controls. The effect seems related to the decreased water intake of the treated animals. The volume differences were less evident during the second half of the study. No other treatment-related effects were observed.

Organ weights:
At termination of exposure, absolute organ weights (most consistently liver weight) were lower in chlorhexidine-treated groups at >= 25 mg/kg bw (groups III, IV, and V) compared to the control group, whereas relative organ weights (esp. kidneys, gonads, brain) were increased.

Gross and histopathology:
Macroscopic findings: no treatment-related effects
Histopathology:
Mesenteric lymph nodes: >= 5 mg/kg bw: histiocytosis with histiocyte conglomerates in the cortical and paracortical areas. Histiocytes showed foamy cytoplasm. In some lymph nodes giant cell formation was observed. There was no marked dose response, but histiocytosis was not observed in the majority of control animals. Histiocytosis was reversible after cessation of treatment but histiocyte conglomerates were still visible at the highest dose after 6 weeks of recovery.
Other lymph nodes (axillary, submaxillary, iliac, thymic) showed no histiocytosis.
Lung: no treatment-related effects but a high incidence of infectious respiratory disease with lymphoreticular proliferation, pneumonia, and bronchiectasis occurred in all groups.
Other organs: no treatment-related effects

Tissue contents of chlorhexidine:
The tissue and blood levels increased with dose and declined after cessation of exposure during the recovery period. Chlorhexidine was detected after 3 months in tissues assayed except for the brain which did not show increased levels up to the highest dose until 18 months. Throughout the study, levels were lowest in brain. The highest levels were found in the mesenteric lymph nodes and in the kidney. The levels in tissue and blood decreased after cessation of exposure. However, after 9 weeks of recovery, high amounts were still detected in the kidneys, and the concentration in the mesenteric lymph nodes was still about 25 % of the concentration found immediately at the end of the exposure period after 18 months.

Effect levels

Dose descriptor:
LOAEL
Effect level:
8.88 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Histiocytosis with histiocyte conglomerates in mesenteric lymph nodes

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The finding of a reactive, non-progressive, reversible histiocytosis in the mesenteric lymph nodes is most probably the result of a local effect in the intestine from the uptake of the compound. There was no evidence for a systemic substance-related effect.
Executive summary:

Chlorhexidine digluconate was administered in drinking water to male and female rats for two years. Recovery groups were included to investigate potential reversibility of effects.

Under the conditions of the study, chlorhexidine digluconate caused histiocytosis of the mesenteric lymph nodes. The appearance of histiocytes was restricted to the cortical and paracortical areas of the node. The effect was reversible after cessation of exposure. There was no evidence of related damage to the intestinal mucosa or the submucosa which could be related to the histiocytosis in the mesenteric lymph node. The histiocytic response in the mesenteric lymph nodes may be explained by: 1.) draining of histiocytes into the node via afferent lymphatics or 2.) an increased production of histiocytes which are normally present in the lymph node as a response to a chemical stimulus.

By the first mode, histiocytes are expected to appear predominantly in the subcapsular regions of the lymph node. However, in the present study, the appearance of histiocytes was restricted essentially to the paracortical and cortical areas of the node. It is considered that the histiocytosis in chlorhexidine digluconate treated rats is a reactive change, probably brought about by the compound entering the nodes via lacteal vessels in a particulate form. Therefore, the proliferation of histiocytes is regarded as a local reaction which is restricted to that node and results from the presence of foreign bodies in the node.

Due to the reduced palatability of the drinking water at higher doses of chlorhexidine digluconate (>= 25 mg/kg bw/d), the animals suffered from dehydration with decreased body weight gain and lowered absolute organ weights compared to controls. No gross or histopathologic lesions were seen in organs except the mesenteric lymph nodes. There were no treatment-related effects in haematology, clinical chemistry and urine analysis.

From this study a LOAEL of 8.88 mg chlorhexidine digluconate/kg bw/d was derived.

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